Human alpha-galactosidase variants

ABSTRACT

The present invention provides engineered human alpha-galactosidase polypeptides and compositions thereof. The engineered human alpha-galactosidase polypeptides have been optimized to provide improved stability under both acidic (pH&lt;4.5) and basic (pH&gt;7) conditions. The invention also relates to the use of the compositions comprising the engineered human alpha-galactosidase polypeptides for therapeutic purposes.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.16/985,742, filed Aug. 5, 2020, which is a continuation of U.S.application Ser. No. 15/529,383, filed May 24, 2017, which is a nationalstage application filed under 35 USC § 371 of International ApplicationNo. PCT/US2015/063329, filed Dec. 2, 2015, which claims the benefitunder 35 U.S.C. § 119(e) of U.S. provisional application Ser. No.62/095,313, filed Dec. 22, 2014, and U.S. provisional application Ser.No. 62/216,452, filed Sep. 10, 2015. The contents of each of the citedapplications are incorporated herein by reference in their entiretiesfor all purposes.

FIELD OF THE INVENTION

The present invention provides engineered human alpha-galactosidasepolypeptides and compositions thereof. The engineered humanalpha-galactosidase polypeptides have been optimized to provide improvedstability under both acidic (pH<4.5) and basic (pH>7) conditions. Theinvention also relates to the use of the compositions comprising theengineered human alpha-galactosidase polypeptides for therapeuticpurposes.

REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

The official copy of the Sequence Listing is submitted concurrently withthe specification as an ASCII formatted text file via EFS-Web, with afile name of “CX7-147WO2UC2_ST25.txt”, a creation date of Mar. 9, 2021,and a size of 2,545,839 bytes. The Sequence Listing filed via EFS-Web ispart of the specification and is incorporated in its entirety byreference herein.

BACKGROUND OF THE INVENTION

Human alpha galactosidase (“GLA”; EC 3.2.1.22) is a lysosomalglycoprotein responsible for hydrolyzing terminal alpha galactosylmoieties from glycolipids and glycoproteins. It works on many substratespresent in a range of human tissues. Fabry disease (also referred to asangiokeratoma corporis diffusum, Anderson-Fabry disease, hereditarydystopic lipidosis, alpha-galactosidase A deficiency, GLA deficiency,and ceramide trihexosidase deficiency) is an X-linked inborn error ofglycosphingolipid catabolism that results from deficient or absentactivity of alpha-galactosidase A. Patients affected with Fabry diseaseaccumulate globotriosylceramide (Gb₃) and related glycosphingolipids inthe plasma and cellular lysosomes of blood vessels, tissue and organs(See e.g., Nance et al., Arch. Neurol., 63:453-457 [2006]). As thepatient ages, the blood vessels become progressively narrowed, due tothe accumulation of these lipids, resulting in decreased blood flow andnourishment to the tissues, particularly in the skin, kidneys, heart,brain, and nervous system. Thus, Fabry disease is a systemic disorderthat manifests as renal failure, cardiac disease, cerebrovasculardisease, small-fiber peripheral neuropathy, and skin lesions, as well asother disorders (See e.g., Schiffmann, Pharm. Ther., 122:65-77 [2009]).Affected patients exhibit symptoms such as painful hands and feet,clusters of small, dark red spots on their skin, the decreased abilityto sweat, corneal opacity, gastrointestinal issues, tinnitus, andhearing loss. Potentially life-threatening complications includeprogressive renal damage, heart attacks, and stroke. This diseaseaffects an estimated 1 in 40,000-60,000 males, but also occurs infemales. Indeed, heterozygous women with Fabry disease experiencesignificant life-threatening conditions requiring medical treatment,including nervous system abnormalities, chronic pain, fatigue, highblood pressure, heart disease, kidney failure, and stroke (See e.g.,Want et al., Genet. Med., 13:457-484 [2011]). Signs of Fabry disease canstart any time from infancy on, with signs usually beginning to showbetween ages 4 and 8, although some patients exhibit a milder,late-onset disease. Treatment is generally supportive and there is nocure for Fabry disease, thus there remains a need for a safe andeffective treatment.

SUMMARY OF THE INVENTION

The present invention provides engineered human alpha-galactosidasepolypeptides and compositions thereof. The engineered humanalpha-galactosidase polypeptides have been optimized to provide improvedstability under both acidic (pH<4.5) and basic (pH>7) conditions. Theinvention also relates to the use of the compositions comprising theengineered human alpha-galactosidase polypeptides for therapeuticpurposes.

The present invention provides recombinant alpha galactosidase A and/orbiologically active recombinant alpha galactosidase A fragmentcomprising an amino acid sequence comprising at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99% sequence identity to SEQID NO:5. In some embodiments, the alpha galactosidase A comprises atleast one mutation in at least one position as provided in Tables 2.1,2.2, 2.4, and/or 2.5, wherein the positions are numbered with referenceto SEQ ID NO:5. In some embodiments, the alpha galactosidase A comprisesat least one mutation in at least one position as provided in Table 2.3,wherein the positions are numbered with reference to SEQ ID NO:10. Insome additional embodiments, the recombinant alpha galactosidase A isderived from a human alpha galactosidase A. In some further embodiments,the recombinant alpha galactosidase A comprises the polypeptide sequenceof SEQ ID NO:15, 13, 10, or 18. In still some additional embodiments,the recombinant alpha galactosidase A is more thermostable than thealpha galactosidase A of SEQ ID NO:5. In some further embodiments, therecombinant alpha galactosidase A is more stable at pH 7.4 than thealpha galactosidase A of SEQ ID NO:5, while in additional embodiments,the recombinant alpha galactosidase A is more stable at pH 4.3 than thealpha galactosidase A of SEQ ID NO:5. In some embodiments therecombinant alpha galactosidase A is more stable at pH 7.4 and pH 4.3than the alpha galactosidase A of SEQ ID NO:5. In still some furtherembodiments, the recombinant alpha galactosidase A is a deimmunizedalpha galactosidase A. In some embodiments, the recombinant alphagalactosidase A is a deimmunized alpha galactosidase A provided in Table7.1. In still some additional embodiments, the recombinant alphagalactosidase A is purified. In some embodiments, the recombinant alphagalactosidase A exhibits at least one improved property selected from:i) enhanced catalytic activity; ii) increased tolerance to pH 7.4; iii)increased tolerance to pH 4.3; or iv) reduced immunogenicity; or acombination of any of i), ii), iii), or iv), as compared to a referencesequence. In some embodiments, the reference sequence is SEQ ID NO:5,while in some alternative embodiments, the reference sequence is SEQ IDNO:10.

The present invention also provides recombinant polynucleotide sequencesencoding at least one recombinant alpha galactosidase A as providedherein (e.g., Tables 2.1, 2.2, 2.3, 2.4, 2.5, and/or Table 7.1). In someembodiments, the recombinant polynucleotide sequence is codon-optimized.

The present invention also provides expression vectors comprising therecombinant polynucleotide sequence encoding at least one recombinantalpha galactosidase A as provided herein (e.g., Tables 2.1, 2.2, 2.3,2.4, 2.5, and/or Table 7.1). In some embodiments, the recombinantpolynucleotide sequence is operably linked to a control sequence. Insome additional embodiments, the control sequence is a promoter. In somefurther embodiments, the promoter is a heterologous promoter. In someembodiments, the expression vector further comprises a signal sequence,as provided herein.

The present invention also provides host cells comprising at least oneexpression vector as provided herein. In some embodiments, the host cellcomprises an expression vector comprising the recombinant polynucleotidesequence encoding at least one recombinant alpha galactosidase A asprovided herein (e.g., Tables 2.1, 2.2, 2.3, 2.4, 2.5, and/or Table7.1). In some embodiments, the host cell is eukaryotic.

The present invention also provides methods of producing an alphagalactosidase A variant, comprising culturing a host cell providedherein, under conditions that the alpha galactosidase A encoded by therecombinant polynucleotide is produced. In some embodiments, the methodsfurther comprise the step of recovering alpha galactosidase A. In somefurther embodiments, the methods further comprise the step of purifyingthe alpha galactosidase A.

The present invention also provides compositions comprising at least onerecombinant alpha galactosidase A as provided herein (e.g., Tables 2.1,2.2, 2.3, 2.4, 2.5, and/or Table 7.1). In some embodiments, the presentinvention provides pharmaceutical compositions. In some additionalembodiments, the present invention provides pharmaceutical compositionsfor the treatment of Fabry disease, comprising an enzyme compositionprovided herein. In some embodiments, the pharmaceutical compositions,further comprise a pharmaceutically acceptable carrier and/or excipient.In some additional embodiments, the pharmaceutical composition issuitable for parenteral injection or infusion to a human.

The present invention also provides methods for treating and/orpreventing the symptoms of Fabry disease in a subject, comprisingproviding a subject having Fabry disease, and providing at least onepharmaceutical composition compositions comprising at least onerecombinant alpha galactosidase A as provided herein (e.g., Tables 2.1,2.2, 2.3, 2.4, 2.5, and/or Table 7.1), and administering thepharmaceutical composition to the subject. In some embodiments, thesymptoms of Fabry disease are ameliorated in the subject. In someadditional embodiments, the subject to whom the pharmaceuticalcomposition of the present invention has been administered is able toeat a diet that is less restricted in its fat content than dietsrequired by subjects exhibiting the symptoms of Fabry disease. In someembodiments, the subject is an infant or child, while in somealternative embodiments, the subject is an adult or young adult.

The present invention also provides for the use of the compositionsprovided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a graph showing the relative activity of different GLAconstructs in S. cerevisiae after 2-5 days of culturing.

FIG. 2 provides graphs showing the Absolute (Panel A) and relative(Panel B) activity of GLA variants after incubation at various pHs.

FIG. 3 provides graphs showing the absolute (Panel A) and relative(Panel B) activity of GLA variants after incubation at varioustemperatures.

FIG. 4 provides graphs showing the absolute (Panel A&B) and relative(Panel C&D) activity of GLA variants after challenge with buffers thatcontain increasing amounts of serum.

FIG. 5 provides a graph showing the relative activity of GLA variantsexpressed in HEK293Tcells.

FIG. 6 provides graphs showing the absolute (Panel A) and relative(Panel B) activity of GLA variants expressed in HEK293T cells,normalized for activity, and incubated at various pHs.

FIG. 7 provides graphs showing the absolute (Panel A) and relative(Panel B) activity of GLA variants expressed in HEK293T cells,normalized for activity, and incubated at various temperatures.

FIG. 8 provides graphs showing GLA variant activity remaining afterincubation in acidic (Panel A) or basic (Panel B) solutions.

FIG. 9 provides a graph showing the GLA activity recovered in rat serumfollowing administration of GLA variants.

DESCRIPTION OF THE INVENTION

The present invention provides engineered human alpha-galactosidasepolypeptides and compositions thereof. The engineered humanalpha-galactosidase polypeptides have been optimized to provide improvedstability under both acidic (pH<4.5) and basic (pH>7) conditions. Theinvention also relates to the use of the compositions comprising theengineered human alpha-galactosidase polypeptides for therapeuticpurposes.

In some embodiments, the engineered human alpha-galactosidasepolypeptides have been optimized to provide improved stability atvarious levels. The invention also relates to the use of thecompositions comprising the engineered human alpha-galactosidasepolypeptides for therapeutic purposes.

Enzyme replacement therapy for treatment of Fabry disease (e.g.,Fabrazyme® agalsidase beta; Genzyme) is available and is considered foreligible individuals. Currently used enzyme replacements therapies arerecombinantly expressed forms of the wild-type human GLA. It is knownthat intravenously administered GLA circulates, becomes endocytosed, andtravels to the endosomes/lysosomes of target organs, where it reducesthe accumulation of Gb3. These drugs do not completely relieve patientsymptoms, as neuropathic pain and transient ischemic attacks continue tooccur at reduced rates. In addition, the uptake of GLA by most targetorgans is poor in comparison to the liver, and the enzyme is unstable atthe pH of blood and lysosomes. Thus, issues remain with availabletreatments. In addition, patients may develop an immune response (IgGand IgE antibodies targeting the administered drug), and suffer severeallergic (anaphylactic) reactions, severe infusion reactions, and evendeath. The present invention is intended to provide more stable enzymessuitable for treatment of Fabry disease, yet with reduced side effectsand improved outcomes, as compared to currently available treatments.Indeed, the present invention is intended to provide recombinant GLAenzymes that have increased stability in blood (pH 7.4), which theenzyme encounters upon injection into the bloodstream. In addition, theenzyme has increased stability at the pH of the lysosome (pH 4.3), thelocation where the enzyme is active during therapy. Thus, directedevolution of recombinantly expressed human GLA in Saccharomycescerevisiae, employing high throughput screening of diverse enzymevariant libraries, was used to provide novel GLA variants with desiredstability properties. In addition, variant enzymes were screened andtheir amino acid sequence determined in order to identify novel GLAvariants with a predicted reduced immunogenicity. By providing GLAvariants with increased pH stability and reduced immunogenicity, thepresent invention provides compositions and methods suitable for use inpatients by increasing patient tolerance of treatment and providingflexibility in dosing and formulation for improved patient outcomes.

Abbreviations and Definitions

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which this invention pertains. Generally,the nomenclature used herein and the laboratory procedures of cellculture, molecular genetics, microbiology, biochemistry, organicchemistry, analytical chemistry and nucleic acid chemistry describedbelow are those well-known and commonly employed in the art. Suchtechniques are well-known and described in numerous texts and referenceworks well known to those of skill in the art. Standard techniques, ormodifications thereof, are used for chemical syntheses and chemicalanalyses. All patents, patent applications, articles and publicationsmentioned herein, both supra and infra, are hereby expresslyincorporated herein by reference.

Although any suitable methods and materials similar or equivalent tothose described herein find use in the practice of the presentinvention, some methods and materials are described herein. It is to beunderstood that this invention is not limited to the particularmethodology, protocols, and reagents described, as these may vary,depending upon the context they are used by those of skill in the art.Accordingly, the terms defined immediately below are more fullydescribed by reference to the application as a whole. All patents,patent applications, articles and publications mentioned herein, bothsupra and infra, are hereby expressly incorporated herein by reference.

Also, as used herein, the singular “a”, “an,” and “the” include theplural references, unless the context clearly indicates otherwise.

Numeric ranges are inclusive of the numbers defining the range. Thus,every numerical range disclosed herein is intended to encompass everynarrower numerical range that falls within such broader numerical range,as if such narrower numerical ranges were all expressly written herein.It is also intended that every maximum (or minimum) numerical limitationdisclosed herein includes every lower (or higher) numerical limitation,as if such lower (or higher) numerical limitations were expresslywritten herein.

The term “about” means an acceptable error for a particular value. Insome instances “about” means within 0.05%, 0.5%, 1.0%, or 2.0%, of agiven value range. In some instances, “about” means within 1, 2, 3, or 4standard deviations of a given value.

Furthermore, the headings provided herein are not limitations of thevarious aspects or embodiments of the invention which can be had byreference to the application as a whole. Accordingly, the terms definedimmediately below are more fully defined by reference to the applicationas a whole. Nonetheless, in order to facilitate understanding of theinvention, a number of terms are defined below.

Unless otherwise indicated, nucleic acids are written left to right in5′ to 3′ orientation; amino acid sequences are written left to right inamino to carboxy orientation, respectively.

As used herein, the term “comprising” and its cognates are used in theirinclusive sense (i.e., equivalent to the term “including” and itscorresponding cognates).

“EC” number refers to the Enzyme Nomenclature of the NomenclatureCommittee of the International Union of Biochemistry and MolecularBiology (NC-IUBMB). The IUBMB biochemical classification is a numericalclassification system for enzymes based on the chemical reactions theycatalyze.

“ATCC” refers to the American Type Culture Collection whosebiorepository collection includes genes and strains.

“NCBI” refers to National Center for Biological Information and thesequence databases provided therein.

“Protein,” “polypeptide,” and “peptide” are used interchangeably hereinto denote a polymer of at least two amino acids covalently linked by anamide bond, regardless of length or post-translational modification(e.g., glycosylation or phosphorylation).

“Amino acids” are referred to herein by either their commonly knownthree-letter symbols or by the one-letter symbols recommended byIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single letter codes.

The term “engineered,” “recombinant,” “non-naturally occurring,” and“variant,” when used with reference to a cell, a polynucleotide or apolypeptide refers to a material or a material corresponding to thenatural or native form of the material that has been modified in amanner that would not otherwise exist in nature or is identical theretobut produced or derived from synthetic materials and/or by manipulationusing recombinant techniques.

As used herein, “wild-type” and “naturally-occurring” refer to the formfound in nature. For example a wild-type polypeptide or polynucleotidesequence is a sequence present in an organism that can be isolated froma source in nature and which has not been intentionally modified byhuman manipulation.

“Deimmunized” as used herein, refers to the manipulation of a proteinsequence to create a variant that is predicted to be not as immunogenicas the wild-type or reference protein. In some embodiments, thepredicted deimmunization is complete, in that the variant protein ispredicted to not stimulate an immune response in patients to whom thevariant protein is administered. This response can be measured byvarious methods including but not limited to, the presence or abundanceof anti-drug antibodies, the presence or abundance of neutralizingantibodies, the presence of an anaphylactic response, peptidepresentation on major histocompatibility complex-II (MHC-II) proteins,or the prevalence or intensity of cytokine release upon administrationof the protein. In some embodiments, the variant protein is lessimmunogenic than the wild-type or reference protein. In someembodiments, deimmunization involves modifications to subsequences ofproteins (e.g., epitopes) that are recognized by human leukocyte antigen(HLA) receptors. In some embodiments, these epitopes are removed bychanging their amino acid sequences to produce a deimmunized variantprotein in which such subsequences are no longer recognized by the HLAreceptors. In some other embodiments, these epitopes retain bindingaffinity to HLA receptors, but are not presented. In some embodiments,the deimmunized protein shows lower levels of response in biochemicaland cell-biological predictors of human immunological responsesincluding dendritic-cell T-cell activation assays, or (HLA) peptidebinding assays. In some embodiments, these epitopes are removed bychanging their amino acid sequence to produce a deimmunized variantprotein in which the epitopes are no longer recognized by T-cellreceptors. In still other embodiments the deimmunized protein inducesanergy in its corresponding T-cells, activates T regulatory cells, orresults in clonal deletion of recognizing B-cells.

“Coding sequence” refers to that part of a nucleic acid (e.g., a gene)that encodes an amino acid sequence of a protein.

The term “percent (%) sequence identity” is used herein to refer tocomparisons among polynucleotides and polypeptides, and are determinedby comparing two optimally aligned sequences over a comparison window,wherein the portion of the polynucleotide or polypeptide sequence in thecomparison window may comprise additions or deletions (i.e., gaps) ascompared to the reference sequence for optimal alignment of the twosequences. The percentage may be calculated by determining the number ofpositions at which the identical nucleic acid base or amino acid residueoccurs in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the window of comparison and multiplying the result by 100to yield the percentage of sequence identity. Alternatively, thepercentage may be calculated by determining the number of positions atwhich either the identical nucleic acid base or amino acid residueoccurs in both sequences or a nucleic acid base or amino acid residue isaligned with a gap to yield the number of matched positions, dividingthe number of matched positions by the total number of positions in thewindow of comparison and multiplying the result by 100 to yield thepercentage of sequence identity. Those of skill in the art appreciatethat there are many established algorithms available to align twosequences. Optimal alignment of sequences for comparison can beconducted, e.g., by the local homology algorithm of Smith and Waterman(Smith and Waterman, Adv. Appl. Math., 2:482 [1981]), by the homologyalignment algorithm of Needleman and Wunsch (Needleman and Wunsch, J.Mol. Biol., 48:443 [1970), by the search for similarity method ofPearson and Lipman (Pearson and Lipman, Proc. Natl. Acad. Sci. USA85:2444 [1988]), by computerized implementations of these algorithms(e.g., GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin SoftwarePackage), or by visual inspection, as known in the art. Examples ofalgorithms that are suitable for determining percent sequence identityand sequence similarity include, but are not limited to the BLAST andBLAST 2.0 algorithms, which are described by Altschul et al. (See,Altschul et al., J. Mol. Biol., 215: 403-410 [1990]; and Altschul etal., 1977, Nucleic Acids Res., 3389-3402 [1977], respectively). Softwarefor performing BLAST analyses is publicly available through the NationalCenter for Biotechnology Information website. This algorithm involvesfirst identifying high scoring sequence pairs (HSPs) by identifyingshort words of length W in the query sequence, which either match orsatisfy some positive-valued threshold score T when aligned with a wordof the same length in a database sequence. T is referred to as, theneighborhood word score threshold (See, Altschul et al, supra). Theseinitial neighborhood word hits act as seeds for initiating searches tofind longer HSPs containing them. The word hits are then extended inboth directions along each sequence for as far as the cumulativealignment score can be increased. Cumulative scores are calculatedusing, for nucleotide sequences, the parameters M (reward score for apair of matching residues; always >0) and N (penalty score formismatching residues; always <0). For amino acid sequences, a scoringmatrix is used to calculate the cumulative score. Extension of the wordhits in each direction are halted when: the cumulative alignment scorefalls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T, and Xdetermine the sensitivity and speed of the alignment. The BLASTN program(for nucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4, and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlength(W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix(See, Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915[1989]). Exemplary determination of sequence alignment and % sequenceidentity can employ the BESTFIT or GAP programs in the GCG WisconsinSoftware package (Accelrys, Madison Wis.), using default parametersprovided.

“Reference sequence” refers to a defined sequence used as a basis for asequence comparison. A reference sequence may be a subset of a largersequence, for example, a segment of a full-length gene or polypeptidesequence. Generally, a reference sequence is at least 20 nucleotide oramino acid residues in length, at least 25 residues in length, at least50 residues in length, at least 100 residues in length or the fulllength of the nucleic acid or polypeptide. Since two polynucleotides orpolypeptides may each (1) comprise a sequence (i.e., a portion of thecomplete sequence) that is similar between the two sequences, and (2)may further comprise a sequence that is divergent between the twosequences, sequence comparisons between two (or more) polynucleotides orpolypeptide are typically performed by comparing sequences of the twopolynucleotides or polypeptides over a “comparison window” to identifyand compare local regions of sequence similarity. In some embodiments, a“reference sequence” can be based on a primary amino acid sequence,where the reference sequence is a sequence that can have one or morechanges in the primary sequence. “Comparison window” refers to aconceptual segment of at least about 20 contiguous nucleotide positionsor amino acids residues wherein a sequence may be compared to areference sequence of at least 20 contiguous nucleotides or amino acidsand wherein the portion of the sequence in the comparison window maycomprise additions or deletions (i.e., gaps) of 20 percent or less ascompared to the reference sequence (which does not comprise additions ordeletions) for optimal alignment of the two sequences. The comparisonwindow can be longer than 20 contiguous residues, and includes,optionally 30, 40, 50, 100, or longer windows.

“Corresponding to”, “reference to” or “relative to” when used in thecontext of the numbering of a given amino acid or polynucleotidesequence refers to the numbering of the residues of a specifiedreference sequence when the given amino acid or polynucleotide sequenceis compared to the reference sequence. In other words, the residuenumber or residue position of a given polymer is designated with respectto the reference sequence rather than by the actual numerical positionof the residue within the given amino acid or polynucleotide sequence.For example, a given amino acid sequence, such as that of an engineeredGLA, can be aligned to a reference sequence by introducing gaps tooptimize residue matches between the two sequences. In these cases,although the gaps are present, the numbering of the residue in the givenamino acid or polynucleotide sequence is made with respect to thereference sequence to which it has been aligned.

“Amino acid difference” or “residue difference” refers to a differencein the amino acid residue at a position of a polypeptide sequencerelative to the amino acid residue at a corresponding position in areference sequence. The positions of amino acid differences generallyare referred to herein as “Xn,” where n refers to the correspondingposition in the reference sequence upon which the residue difference isbased. For example, a “residue difference at position X93 as compared toSEQ ID NO:2” refers to a difference of the amino acid residue at thepolypeptide position corresponding to position 93 of SEQ ID NO:2. Thus,if the reference polypeptide of SEQ ID NO:2 has a serine at position 93,then a “residue difference at position X93 as compared to SEQ ID NO:2”an amino acid substitution of any residue other than serine at theposition of the polypeptide corresponding to position 93 of SEQ ID NO:2.In most instances herein, the specific amino acid residue difference ata position is indicated as “XnY” where “Xn” specified the correspondingposition as described above, and “Y” is the single letter identifier ofthe amino acid found in the engineered polypeptide (i.e., the differentresidue than in the reference polypeptide). In some instances (e.g., inTables 2.1, 2.2, 2.3, 2.4, 2.5, and 6.1), the present disclosure alsoprovides specific amino acid differences denoted by the conventionalnotation “AnB”, where A is the single letter identifier of the residuein the reference sequence, “n” is the number of the residue position inthe reference sequence, and B is the single letter identifier of theresidue substitution in the sequence of the engineered polypeptide. Insome instances, a polypeptide of the present disclosure can include oneor more amino acid residue differences relative to a reference sequence,which is indicated by a list of the specified positions where residuedifferences are present relative to the reference sequence. In someembodiments, where more than one amino acid can be used in a specificresidue position of a polypeptide, the various amino acid residues thatcan be used are separated by a “/” (e.g., X307H/X307P or X307H/P). Insome embodiments, the enzyme variants comprise more than onesubstitution. These substitutions are separated by a slash for ease inreading (e.g., C143A/K206A). The present application includes engineeredpolypeptide sequences comprising one or more amino acid differences thatinclude either/or both conservative and non-conservative amino acidsubstitutions.

“Conservative amino acid substitution” refers to a substitution of aresidue with a different residue having a similar side chain, and thustypically involves substitution of the amino acid in the polypeptidewith amino acids within the same or similar defined class of aminoacids. By way of example and not limitation, an amino acid with analiphatic side chain may be substituted with another aliphatic aminoacid (e.g., alanine, valine, leucine, and isoleucine); an amino acidwith hydroxyl side chain is substituted with another amino acid with ahydroxyl side chain (e.g., serine and threonine); an amino acids havingaromatic side chains is substituted with another amino acid having anaromatic side chain (e.g., phenylalanine, tyrosine, tryptophan, andhistidine); an amino acid with a basic side chain is substituted withanother amino acid with a basis side chain (e.g., lysine and arginine);an amino acid with an acidic side chain is substituted with anotheramino acid with an acidic side chain (e.g., aspartic acid or glutamicacid); and/or a hydrophobic or hydrophilic amino acid is replaced withanother hydrophobic or hydrophilic amino acid, respectively.

“Non-conservative substitution” refers to substitution of an amino acidin the polypeptide with an amino acid with significantly differing sidechain properties. Non-conservative substitutions may use amino acidsbetween, rather than within, the defined groups and affects (a) thestructure of the peptide backbone in the area of the substitution (e.g.,proline for glycine) (b) the charge or hydrophobicity, or (c) the bulkof the side chain. By way of example and not limitation, an exemplarynon-conservative substitution can be an acidic amino acid substitutedwith a basic or aliphatic amino acid; an aromatic amino acid substitutedwith a small amino acid; and a hydrophilic amino acid substituted with ahydrophobic amino acid.

“Deletion” refers to modification to the polypeptide by removal of oneor more amino acids from the reference polypeptide. Deletions cancomprise removal of 1 or more amino acids, 2 or more amino acids, 5 ormore amino acids, 10 or more amino acids, 15 or more amino acids, or 20or more amino acids, up to 10% of the total number of amino acids, or upto 20% of the total number of amino acids making up the reference enzymewhile retaining enzymatic activity and/or retaining the improvedproperties of an engineered enzyme. Deletions can be directed to theinternal portions and/or terminal portions of the polypeptide. Invarious embodiments, the deletion can comprise a continuous segment orcan be discontinuous.

“Insertion” refers to modification to the polypeptide by addition of oneor more amino acids from the reference polypeptide. Insertions can be inthe internal portions of the polypeptide, or to the carboxy or aminoterminus. Insertions as used herein include fusion proteins as is knownin the art. The insertion can be a contiguous segment of amino acids orseparated by one or more of the amino acids in the naturally occurringpolypeptide.

A “functional fragment” or a “biologically active fragment” usedinterchangeably herein refers to a polypeptide that has anamino-terminal and/or carboxy-terminal deletion(s) and/or internaldeletions, but where the remaining amino acid sequence is identical tothe corresponding positions in the sequence to which it is beingcompared (e.g., a full-length engineered GLA of the present invention)and that retains substantially all of the activity of the full-lengthpolypeptide.

“Isolated polypeptide” refers to a polypeptide which is substantiallyseparated from other contaminants that naturally accompany it, e.g.,protein, lipids, and polynucleotides. The term embraces polypeptideswhich have been removed or purified from their naturally-occurringenvironment or expression system (e.g., host cell or in vitrosynthesis). The recombinant GLA polypeptides may be present within acell, present in the cellular medium, or prepared in various forms, suchas lysates or isolated preparations. As such, in some embodiments, therecombinant GLA polypeptides can be an isolated polypeptide.

“Substantially pure polypeptide” refers to a composition in which thepolypeptide species is the predominant species present (i.e., on a molaror weight basis it is more abundant than any other individualmacromolecular species in the composition), and is generally asubstantially purified composition when the object species comprises atleast about 50 percent of the macromolecular species present by mole or% weight. Generally, a substantially pure GLA composition comprisesabout 60% or more, about 70% or more, about 80% or more, about 90% ormore, about 95% or more, and about 98% or more of all macromolecularspecies by mole or % weight present in the composition. In someembodiments, the object species is purified to essential homogeneity(i.e., contaminant species cannot be detected in the composition byconventional detection methods) wherein the composition consistsessentially of a single macromolecular species. Solvent species, smallmolecules (<500 Daltons), and elemental ion species are not consideredmacromolecular species. In some embodiments, the isolated recombinantGLA polypeptides are substantially pure polypeptide compositions.

“Improved enzyme property” refers to an engineered GLA polypeptide thatexhibits an improvement in any enzyme property as compared to areference GLA polypeptide and/or as a wild-type GLA polypeptide oranother engineered GLA polypeptide. Improved properties include but arenot limited to such properties as increased protein expression,increased thermoactivity, increased thermostability, increased pHactivity, increased stability, increased enzymatic activity, increasedsubstrate specificity or affinity, increased specific activity,increased resistance to substrate or end-product inhibition, increasedchemical stability, improved chemoselectivity, improved solventstability, increased tolerance to acidic or basic pH, increasedtolerance to proteolytic activity (i.e., reduced sensitivity toproteolysis), reduced aggregation, increased solubility, reducedimmunogenicity, improved post-translational modification (e.g.,glycosylation), and altered temperature profile.

“Increased enzymatic activity” or “enhanced catalytic activity” refersto an improved property of the engineered GLA polypeptides, which can berepresented by an increase in specific activity (e.g., productproduced/time/weight protein) or an increase in percent conversion ofthe substrate to the product (e.g., percent conversion of startingamount of substrate to product in a specified time period using aspecified amount of GLA) as compared to the reference GLA enzyme.Exemplary methods to determine enzyme activity are provided in theExamples. Any property relating to enzyme activity may be affected,including the classical enzyme properties of K_(m), V_(max) or k_(cat),changes of which can lead to increased enzymatic activity. Improvementsin enzyme activity can be from about 1.1 fold the enzymatic activity ofthe corresponding wild-type enzyme, to as much as 2-fold, 5-fold,10-fold, 20-fold, 25-fold, 50-fold, 75-fold, 100-fold, 150-fold,200-fold or more enzymatic activity than the naturally occurring GLA oranother engineered GLA from which the GLA polypeptides were derived.

In some embodiments, the engineered GLA polypeptides have a k_(cat) ofat least 0.1/sec, at least 0.5/sec, at least 1.0/sec, at least 5.0/sec,at least 10.0/sec and in some preferred embodiments greater than10.0/sec. In some embodiments, the K_(m) is in the range of about 1 μMto about 5 mM; in the range of about 5 μM to about 2 mM; in the range ofabout 10 μM to about 2 mM; or in the range of about 10 μM to about 1 mM.In some specific embodiments, the engineered GLA enzyme exhibitsimproved enzymatic activity after exposure to certain conditions in therange of 1.5 to 10 fold, 1.5 to 25 fold, 1.5 to 50 fold, 1.5 to 100 foldor greater than that of a reference GLA enzyme (e.g., a wild-type GLA orany other reference GLA). GLA activity can be measured by any suitablemethod known in the art (e.g., standard assays, such as monitoringchanges in spectrophotometric properties of reactants or products). Insome embodiments, the amount of products produced can be measured byHigh-Performance Liquid Chromatography (HPLC) separation combined withUV absorbance or fluorescent detection directly or followingo-phthaldialdehyde (OPA) derivatization. Comparisons of enzymeactivities are made using a defined preparation of enzyme, a definedassay under a set condition, and one or more defined substrates, asfurther described in detail herein. Generally, when lysates arecompared, the numbers of cells and the amount of protein assayed aredetermined as well as use of identical expression systems and identicalhost cells to minimize variations in amount of enzyme produced by thehost cells and present in the lysates.

The term “improved tolerance to acidic pH” means that a recombinant GLAaccording to the invention will have increased stability (higherretained activity at about pH 4.8 after exposure to acidic pH for aspecified period of time (1 hour, up to 24 hours)) as compared to areference GLA or another enzyme.

“Physiological pH” as used herein means the pH range generally found ina subject's (e.g., human) blood.

The term “basic pH” (e.g., used with reference to improved stability tobasic pH conditions or increased tolerance to basic pH) means a pH rangeof about 7 to 11.

The term “acidic pH” (e.g., used with reference to improved stability toacidic pH conditions or increased tolerance to acidic pH) means a pHrange of about 1.5 to 4.5.

“Conversion” refers to the enzymatic conversion (or biotransformation)of a substrate(s) to the corresponding product(s). “Percent conversion”refers to the percent of the substrate that is converted to the productwithin a period of time under specified conditions. Thus, the “enzymaticactivity” or “activity” of a GLA polypeptide can be expressed as“percent conversion” of the substrate to the product in a specificperiod of time.

“Hybridization stringency” relates to hybridization conditions, such aswashing conditions, in the hybridization of nucleic acids. Generally,hybridization reactions are performed under conditions of lowerstringency, followed by washes of varying but higher stringency. Theterm “moderately stringent hybridization” refers to conditions thatpermit target-DNA to bind a complementary nucleic acid that has about60% identity, preferably about 75% identity, about 85% identity to thetarget DNA, with greater than about 90% identity totarget-polynucleotide. Exemplary moderately stringent conditions areconditions equivalent to hybridization in 50% formamide, 5×Denhart'ssolution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.2×SSPE,0.2% SDS, at 42° C. “High stringency hybridization” refers generally toconditions that are about 10° C. or less from the thermal meltingtemperature T_(m) as determined under the solution condition for adefined polynucleotide sequence. In some embodiments, a high stringencycondition refers to conditions that permit hybridization of only thosenucleic acid sequences that form stable hybrids in 0.018M NaCl at 65° C.(i.e., if a hybrid is not stable in 0.018M NaCl at 65° C., it will notbe stable under high stringency conditions, as contemplated herein).High stringency conditions can be provided, for example, byhybridization in conditions equivalent to 50% formamide, 5×Denhart'ssolution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.1×SSPE,and 0.1% SDS at 65° C. Another high stringency condition is hybridizingin conditions equivalent to hybridizing in 5×SSC containing 0.1% (w:v)SDS at 65° C. and washing in 0.1×SSC containing 0.1% SDS at 65° C. Otherhigh stringency hybridization conditions, as well as moderatelystringent conditions, are described in the references cited above.

“Codon optimized” refers to changes in the codons of the polynucleotideencoding a protein to those preferentially used in a particular organismsuch that the encoded protein is more efficiently expressed in theorganism of interest. Although the genetic code is degenerate in thatmost amino acids are represented by several codons, called “synonyms” or“synonymous” codons, it is well known that codon usage by particularorganisms is nonrandom and biased towards particular codon triplets.This codon usage bias may be higher in reference to a given gene, genesof common function or ancestral origin, highly expressed proteins versuslow copy number proteins, and the aggregate protein coding regions of anorganism's genome. In some embodiments, the polynucleotides encoding theGLA enzymes may be codon optimized for optimal production from the hostorganism selected for expression.

“Control sequence” refers herein to include all components, which arenecessary or advantageous for the expression of a polynucleotide and/orpolypeptide of the present application. Each control sequence may benative or foreign to the nucleic acid sequence encoding the polypeptide.Such control sequences include, but are not limited to, a leader,polyadenylation sequence, propeptide sequence, promoter sequence, signalpeptide sequence, initiation sequence and transcription terminator. At aminimum, the control sequences include a promoter, and transcriptionaland translational stop signals. The control sequences may be providedwith linkers for the purpose of introducing specific restriction sitesfacilitating ligation of the control sequences with the coding region ofthe nucleic acid sequence encoding a polypeptide.

“Operably linked” is defined herein as a configuration in which acontrol sequence is appropriately placed (i.e., in a functionalrelationship) at a position relative to a polynucleotide of interestsuch that the control sequence directs or regulates the expression ofthe polynucleotide and/or polypeptide of interest.

“Promoter sequence” refers to a nucleic acid sequence that is recognizedby a host cell for expression of a polynucleotide of interest, such as acoding sequence. The promoter sequence contains transcriptional controlsequences, which mediate the expression of a polynucleotide of interest.The promoter may be any nucleic acid sequence which showstranscriptional activity in the host cell of choice including mutant,truncated, and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

“Suitable reaction conditions” refers to those conditions in theenzymatic conversion reaction solution (e.g., ranges of enzyme loading,substrate loading, temperature, pH, buffers, co-solvents, etc.) underwhich a GLA polypeptide of the present application is capable ofconverting a substrate to the desired product compound, Exemplary“suitable reaction conditions” are provided in the present applicationand illustrated by the Examples. “Loading”, such as in “compoundloading” or “enzyme loading” refers to the concentration or amount of acomponent in a reaction mixture at the start of the reaction.“Substrate” in the context of an enzymatic conversion reaction processrefers to the compound or molecule acted on by the GLA polypeptide.“Product” in the context of an enzymatic conversion process refers tothe compound or molecule resulting from the action of the GLApolypeptide on a substrate.

As used herein the term “culturing” refers to the growing of apopulation of microbial cells under any suitable conditions (e.g., usinga liquid, gel or solid medium).

Recombinant polypeptides can be produced using any suitable methodsknown the art. Genes encoding the wild-type polypeptide of interest canbe cloned in vectors, such as plasmids, and expressed in desired hosts,such as E. coli, S. cerevisiae, etc. Variants of recombinantpolypeptides can be generated by various methods known in the art.Indeed, there is a wide variety of different mutagenesis techniques wellknown to those skilled in the art. In addition, mutagenesis kits arealso available from many commercial molecular biology suppliers. Methodsare available to make specific substitutions at defined amino acids(site-directed), specific or random mutations in a localized region ofthe gene (regio-specific), or random mutagenesis over the entire gene(e.g., saturation mutagenesis). Numerous suitable methods are known tothose in the art to generate enzyme variants, including but not limitedto site-directed mutagenesis of single-stranded DNA or double-strandedDNA using PCR, cassette mutagenesis, gene synthesis, error-prone PCR,shuffling, and chemical saturation mutagenesis, or any other suitablemethod known in the art. Non-limiting examples of methods used for DNAand protein engineering are provided in the following patents: U.S. Pat.Nos. 6,117,679; 6,420,175; 6,376,246; 6,586,182; 7,747,391; 7,747,393;7,783,428; and 8,383,346. After the variants are produced, they can bescreened for any desired property (e.g., high or increased activity, orlow or reduced activity, increased thermal activity, increased thermalstability, and/or acidic pH stability, etc.). In some embodiments,“recombinant GLA polypeptides” (also referred to herein as “engineeredGLA polypeptides,” “variant GLA enzymes,” and “GLA variants”) find use.

As used herein, a “vector” is a DNA construct for introducing a DNAsequence into a cell. In some embodiments, the vector is an expressionvector that is operably linked to a suitable control sequence capable ofeffecting the expression in a suitable host of the polypeptide encodedin the DNA sequence. In some embodiments, an “expression vector” has apromoter sequence operably linked to the DNA sequence (e.g., transgene)to drive expression in a host cell, and in some embodiments, alsocomprises a transcription terminator sequence.

As used herein, the term “expression” includes any step involved in theproduction of the polypeptide including, but not limited to,transcription, post-transcriptional modification, translation, andpost-translational modification. In some embodiments, the term alsoencompasses secretion of the polypeptide from a cell.

As used herein, the term “produces” refers to the production of proteinsand/or other compounds by cells. It is intended that the term encompassany step involved in the production of polypeptides including, but notlimited to, transcription, post-transcriptional modification,translation, and post-translational modification. In some embodiments,the term also encompasses secretion of the polypeptide from a cell.

As used herein, an amino acid or nucleotide sequence (e.g., a promotersequence, signal peptide, terminator sequence, etc.) is “heterologous”to another sequence with which it is operably linked if the twosequences are not associated in nature.

As used herein, the terms “host cell” and “host strain” refer tosuitable hosts for expression vectors comprising DNA provided herein(e.g., the polynucleotides encoding the GLA variants). In someembodiments, the host cells are prokaryotic or eukaryotic cells thathave been transformed or transfected with vectors constructed usingrecombinant DNA techniques as known in the art.

The term “analogue” means a polypeptide having more than 70% sequenceidentity but less than 100% sequence identity (e.g., more than 75%, 78%,80%, 83%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%sequence identity) with a reference polypeptide. In some embodiments,analogues means polypeptides that contain one or more non-naturallyoccurring amino acid residues including, but not limited, tohomoarginine, ornithine and norvaline, as well as naturally occurringamino acids. In some embodiments, analogues also include one or moreD-amino acid residues and non-peptide linkages between two or more aminoacid residues.

The term “therapeutic” refers to a compound administered to a subjectwho shows signs or symptoms of pathology having beneficial or desirablemedical effects.

The term “pharmaceutical composition” refers to a composition suitablefor pharmaceutical use in a mammalian subject (e.g., human) comprising apharmaceutically effective amount of an engineered GLA polypeptideencompassed by the invention and an acceptable carrier.

The term “effective amount” means an amount sufficient to produce thedesired result. One of general skill in the art may determine what theeffective amount by using routine experimentation.

The terms “isolated” and “purified” are used to refer to a molecule(e.g., an isolated nucleic acid, polypeptide, etc.) or other componentthat is removed from at least one other component with which it isnaturally associated. The term “purified” does not require absolutepurity, rather it is intended as a relative definition.

The term “subject” encompasses mammals such as humans, non-humanprimates, livestock, companion animals, and laboratory animals (e.g.,rodents and lagamorphs). It is intended that the term encompass femalesas well as males.

As used herein, the term “patient” means any subject that is beingassessed for, treated for, or is experiencing disease.

The term “infant” refers to a child in the period of the first monthafter birth to approximately one (1) year of age. As used herein, theterm “newborn” refers to child in the period from birth to the 28^(th)day of life. The term “premature infant” refers to an infant born afterthe twentieth completed week of gestation, yet before full term,generally weighing ˜500 to ˜2499 grams at birth. A “very low birthweight infant” is an infant weighing less than 1500 g at birth.

As used herein, the term “child” refers to a person who has not attainedthe legal age for consent to treatment or research procedures. In someembodiments, the term refers to a person between the time of birth andadolescence.

As used herein, the term “adult” refers to a person who has attainedlegal age for the relevant jurisdiction (e.g., 18 years of age in theUnited States). In some embodiments, the term refers to any fully grown,mature organism. In some embodiments, the term “young adult” refers to aperson less than 18 years of age, but who has reached sexual maturity.

As used herein, “composition” and “formulation” encompass productscomprising at least one engineered GLA of the present invention,intended for any suitable use (e.g., pharmaceutical compositions,dietary/nutritional supplements, feed, etc.).

The terms “administration” and “administering” a composition meanproviding a composition of the present invention to a subject (e.g., toa person suffering from the effects of Fabry disease).

The term “carrier” when used in reference to a pharmaceuticalcomposition means any of the standard pharmaceutical carrier, buffers,and excipients, such as stabilizers, preservatives, and adjuvants.

The term “pharmaceutically acceptable” means a material that can beadministered to a subject without causing any undesirable biologicaleffects or interacting in a deleterious manner with any of thecomponents in which it is contained and that possesses the desiredbiological activity.

As used herein, the term “excipient” refers to any pharmaceuticallyacceptable additive, carrier, diluent, adjuvant, or other ingredient,other than the active pharmaceutical ingredient (API; e.g., theengineered GLA polypeptides of the present invention). Excipients aretypically included for formulation and/or administration purposes.

The term “therapeutically effective amount” when used in reference tosymptoms of disease/condition refers to the amount and/or concentrationof a compound (e.g., engineered GLA polypeptides) that ameliorates,attenuates, or eliminates one or more symptom of a disease/condition orprevents or delays the onset of symptom(s).

The term “therapeutically effective amount” when used in reference to adisease/condition refers to the amount and/or concentration of acomposition (e.g., engineered GLA polypeptides) that ameliorates,attenuates, or eliminates the disease/condition. In some embodiments,the term is use in reference to the amount of a composition that elicitsthe biological (e.g., medical) response by a tissue, system, or animalsubject that is sought by the researcher, physician, veterinarian, orother clinician.

It is intended that the terms “treating,” “treat” and “treatment”encompass preventative (e.g., prophylactic), as well as palliativetreatment.

Engineered GLA Expression and Activity:

Two strategies for secreted GLA expression were utilized, using theyeast MFα signal peptide (MF-SP) or a longer leader sequence of 83 aminoacids (MF-leader) to drive secretion of a yeast codon-optimized maturehuman GLA. Clones were expressed from a pYT-72 vector in S. cerevisiaestrain INVSc1. Both approaches provided supernatants with measurableactivity on the fluorogenic substrate 4-methylumbelliferylα-D-galactopyranoside (4-MuGal). However, the construct with the yeastMFα signal peptide provided 3-fold higher activities and was used as thestarting sequence for directed evolution.

To identify mutational diversity, a 13-position conserved “homolog”combinatorial library and a 192-position site saturation mutagenesislibrary were constructed. Equivalent volumes of supernatant werescreened in an unchallenged condition (no incubation, pH 4.8) orfollowing a one-hour incubation in a low pH (3.9-4.2) or high pH(7.1-8.2) environment. GLA variants with increased activity due toincreased GLA expression or GLA specific activity were identified basedon their fold improvement over the parent GLA. GLA variants withincreased stability were identified by dividing the fold-improvementobserved under challenged conditions by the fold-improvement observedunder unchallenged conditions. This approach reduces the bias towardsselecting variants based on increased expression but without changes inspecific activity at pH extremes. Composite activity scores (the productof fold-improvements for all three conditions) and stability (theproduct of stability scores) were used to rank mutations in improvedvariants for inclusion in subsequent GLA libraries.

Engineered GLA:

In some embodiments the engineered GLA which exhibits an improvedproperty has at least about 85%, at least about 88%, at least about 90%,at least about 91%, at least about 92%, at least about 93%, at leastabout 94%, at least about 95%, at least about 96%, at least about 97%,at least about 98%, at least about 99%, or at about 100% amino acidsequence identity with SEQ ID NO:5, and an amino acid residue differenceas compared to SEQ ID NO:5, at one or more amino acid positions (such asat 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 20 or more amino acidpositions compared to SEQ ID NO:5, or a sequence having at least 85%, atleast 88%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% or greater amino acid sequence identity with SEQ ID NO:5). Insome embodiment the residue difference as compared to SEQ ID NO:5, atone or more positions will include at least 1, 2, 3, 4, 5, 6, 7, 8, 9,10 or more conservative amino acid substitutions. In some embodiments,the engineered GLA polypeptide is a polypeptide listed in Table 2.1,2.2, 2.4, 2.5, or Table 7.1.

In some embodiments the engineered GLA which exhibits an improvedproperty has at least about 85%, at least about 88%, at least about 90%,at least about 91%, at least about 92%, at least about 93%, at leastabout 94%, at least about 95%, at least about 96%, at least about 97%,at least about 98%, at least about 99%, or at about 100% amino acidsequence identity with SEQ ID NO:10, and an amino acid residuedifference as compared to SEQ ID NO:10, at one or more amino acidpositions (such as at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 20or more amino acid positions compared to SEQ ID NO:10, or a sequencehaving at least 85%, at least 88%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99% or greater amino acid sequence identitywith SEQ ID NO:10). In some embodiment the residue difference ascompared to SEQ ID NO:10, at one or more positions will include at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative amino acidsubstitutions. In some embodiments, the engineered GLA polypeptide is apolypeptide listed in Table 2.3.

In some embodiments the engineered GLA which exhibits an improvedproperty has at least 85%, at least 88%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% amino acid sequence identitywith SEQ ID NO:5. In some embodiments the engineered GLA which exhibitsan improved property has at least 85%, at least 88%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% amino acidsequence identity with SEQ ID NO:10.

In some embodiments, the engineered GLA polypeptide is selected from SEQID NOS:15, 13, 10, and 18.

In some embodiments, the engineered GLA polypeptide comprises afunctional fragment of an engineered GLA polypeptide encompassed by theinvention. Functional fragments have at least 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% of the activity of the engineered GLA polypeptide from which is wasderived (i.e., the parent engineered GLA). A functional fragmentcomprises at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and even 99% of the parentsequence of the engineered GLA. In some embodiments the functionalfragment is truncated by less than 5, less than 10, less than 15, lessthan 10, less than 25, less than 30, less than 35, less than 40, lessthan 45, and less than 50 amino acids.

Polynucleotides Encoding Engineered Polypeptides, Expression Vectors andHost Cells:

The present invention provides polynucleotides encoding the engineeredGLA polypeptides described herein. In some embodiments, thepolynucleotides are operatively linked to one or more heterologousregulatory sequences that control gene expression to create arecombinant polynucleotide capable of expressing the polypeptide.Expression constructs containing a heterologous polynucleotide encodingthe engineered GLA polypeptides can be introduced into appropriate hostcells to express the corresponding GLA polypeptide.

As will be apparent to the skilled artisan, availability of a proteinsequence and the knowledge of the codons corresponding to the variousamino acids provide a description of all the polynucleotides capable ofencoding the subject polypeptides. The degeneracy of the genetic code,where the same amino acids are encoded by alternative or synonymouscodons, allows an extremely large number of nucleic acids to be made,all of which encode the engineered GLA polypeptide. Thus, havingknowledge of a particular amino acid sequence, those skilled in the artcould make any number of different nucleic acids by simply modifying thesequence of one or more codons in a way which does not change the aminoacid sequence of the protein. In this regard, the present inventionspecifically contemplates each and every possible variation ofpolynucleotides that could be made encoding the polypeptides describedherein by selecting combinations based on the possible codon choices,and all such variations are to be considered specifically disclosed forany polypeptide described herein, including the variants provided inTables 2.1, 2.2, 2.3, 2.4, 2.5, and 6.1.

In various embodiments, the codons are preferably selected to fit thehost cell in which the protein is being produced. For example, preferredcodons used in bacteria are used for expression in bacteria.Consequently, codon optimized polynucleotides encoding the engineeredGLA polypeptides contain preferred codons at about 40%, 50%, 60%, 70%,80%, or greater than 90% of codon positions of the full length codingregion.

In some embodiments, as described above, the polynucleotide encodes anengineered polypeptide having GLA activity with the properties disclosedherein, wherein the polypeptide comprises an amino acid sequence havingat least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to a referencesequence selected from SEQ ID NOS:5, and/or 10, or the amino acidsequence of any variant as disclosed in Tables 2.1, 2.2, 2.3, 2.4, 2.5,or 6.1, and one or more residue differences as compared to the referencepolypeptide of SEQ ID NOS:5, and/or 10, or the amino acid sequence ofany variant as disclosed in Tables 2.1, 2.2, 2.3, 2.4, 2.5, or 6.1, (forexample 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid residuepositions). In some embodiments, the reference sequence is selected fromSEQ ID NO:5 and/or 10. In some embodiments, the polynucleotide encodesan engineered polypeptide having GLA activity with the propertiesdisclosed herein, wherein the polypeptide comprises an amino acidsequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity to reference sequence SEQ ID NO:5, and one or more residuedifferences as compared to SEQ ID NO:5, at residue positions selectedfrom those provided in Tables 2.1, 2.2, 2.4, 2.5, or 6.1, when optimallyaligned with the polypeptide of SEQ ID NO:5.

In some embodiments, the polynucleotide encodes an engineeredpolypeptide having GLA activity with the properties disclosed herein,wherein the polypeptide comprises an amino acid sequence having at least80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to referencesequence SEQ ID NO:10, and one or more residue differences as comparedto SEQ ID NO:10, at residue positions selected from those provided inTables 2.3, when optimally aligned with the polypeptide of SEQ ID NO:10.

In some embodiments, the polynucleotide encoding the engineered GLApolypeptides comprises a polynucleotide sequence selected from apolynucleotide sequence encoding SEQ ID NOS:10, 13, 15, 18, 21, and 24.In some embodiments, the polynucleotide encoding an engineered GLApolypeptide has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 93%, 95%, 96%, 97%, 98%, 99% nucleotide residue identity toSEQ ID NOS: 8, 9, 11, 12, 14, 16, 17, 19, 20, 22, and/or 23. In someembodiments, the polynucleotide encoding the engineered GLA polypeptidescomprises a polynucleotide sequence selected from SEQ ID NOS:8, 9, 11,12, 14, 16, 17, 19, 20, 22, and 23.

In some embodiments, the polynucleotides are capable of hybridizingunder highly stringent conditions to a reference polynucleotide sequenceselected from SEQ ID NOS: 8, 9, 11, 12, 14, 16, 17, 19, 20, 22, and 23,or a complement thereof, or a polynucleotide sequence encoding any ofthe variant GLA polypeptides provided herein. In some embodiments, thepolynucleotide capable of hybridizing under highly stringent conditionsencodes a GLA polypeptide comprising an amino acid sequence that has oneor more residue differences as compared to SEQ ID NO:5 and/or 10, atresidue positions selected from any positions as set forth in Tables2.1, 2.2, 2.3, 2.4, 2.5, and/or 6.1.

In some embodiments, an isolated polynucleotide encoding any of theengineered GLA polypeptides provided herein is manipulated in a varietyof ways to provide for expression of the polypeptide. In someembodiments, the polynucleotides encoding the polypeptides are providedas expression vectors where one or more control sequences is present toregulate the expression of the polynucleotides and/or polypeptides.Manipulation of the isolated polynucleotide prior to its insertion intoa vector may be desirable or necessary depending on the expressionvector. The techniques for modifying polynucleotides and nucleic acidsequences utilizing recombinant DNA methods are well known in the art.

In some embodiments, the control sequences include among othersequences, promoters, leader sequences, polyadenylation sequences,propeptide sequences, signal peptide sequences, and transcriptionterminators. As known in the art, suitable promoters can be selectedbased on the host cells used. Exemplary promoters for filamentous fungalhost cells, include promoters obtained from the genes for Aspergillusoryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillusniger neutral alpha-amylase, Aspergillus niger acid stablealpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase(glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease,Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulansacetamidase, and Fusarium oxysporum trypsin-like protease (See e.g., WO96/00787), as well as the NA2-tpi promoter (a hybrid of the promotersfrom the genes for Aspergillus niger neutral alpha-amylase andAspergillus oryzae triose phosphate isomerase), and mutant, truncated,and hybrid promoters thereof. Exemplary yeast cell promoters can be fromthe genes can be from the genes for Saccharomyces cerevisiae enolase(ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomycescerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphatedehydrogenase (ADH2/GAP), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare known in the art (See e.g., Romanos et al., Yeast 8:423-488 [1992]).Exemplary promoters for use in mammalian cells include, but are notlimited to those from cytomegalovirus (CMV), Simian vacuolating virus 40(SV40), from Homo sapiens phosphorglycerate kinase, beta actin,elongation factor-1a or glyceraldehyde-3-phosphate dehydrogenase, orfrom Gallus gallus' β-actin.

In some embodiments, the control sequence is a suitable transcriptionterminator sequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleic acid sequence encoding the polypeptide. Anyterminator which is functional in the host cell of choice finds use inthe present invention. For example, exemplary transcription terminatorsfor filamentous fungal host cells can be obtained from the genes forAspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase,Aspergillus nidulans anthranilate synthase, Aspergillus nigeralpha-glucosidase, and Fusarium oxysporum trypsin-like protease.Exemplary terminators for yeast host cells can be obtained from thegenes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are known in the art (See e.g., Romanos et al., supra).Exemplary terminators for mammalian cells include, but are not limitedto those from cytomegalovirus (CMV), Simian vacuolating virus 40 (SV40),or from Homo sapiens growth hormone.

In some embodiments, the control sequence is a suitable leader sequence,a non-translated region of an mRNA that is important for translation bythe host cell. The leader sequence is operably linked to the 5′ terminusof the nucleic acid sequence encoding the polypeptide. Any leadersequence that is functional in the host cell of choice may be used.Exemplary leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase. Suitable leaders for yeast host cellsinclude, but are not limited to those obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′ terminus of the nucleic acid sequence andwhich, when transcribed, is recognized by the host cell as a signal toadd polyadenosine residues to transcribed mRNA. Any polyadenylationsequence which is functional in the host cell of choice may be used inthe present invention. Exemplary polyadenylation sequences forfilamentous fungal host cells include, but are not limited to those fromthe genes for Aspergillus oryzae TAKA amylase, Aspergillus nigerglucoamylase, Aspergillus nidulans anthranilate synthase, Fusariumoxysporum trypsin-like protease, and Aspergillus nigeralpha-glucosidase. Useful polyadenylation sequences for yeast host cellsare also known in the art (See e.g., Guo and Sherman, Mol. Cell. Bio.,15:5983-5990 [1995]).

In some embodiments, the control sequence is a signal peptide codingregion that codes for an amino acid sequence linked to the aminoterminus of a polypeptide and directs the encoded polypeptide into thecell's secretory pathway. The 5′ end of the coding sequence of thenucleic acid sequence may inherently contain a signal peptide codingregion naturally linked in translation reading frame with the segment ofthe coding region that encodes the secreted polypeptide. Alternatively,the 5′ end of the coding sequence may contain a signal peptide codingregion that is foreign to the coding sequence. Any signal peptide codingregion that directs the expressed polypeptide into the secretory pathwayof a host cell of choice finds use for expression of the engineered GLApolypeptides provided herein. Effective signal peptide coding regionsfor filamentous fungal host cells include, but are not limited to thesignal peptide coding regions obtained from the genes for Aspergillusoryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillusniger glucoamylase, Rhizomucor miehei aspartic proteinase, Humicolainsolens cellulase, and Humicola lanuginosa lipase. Useful signalpeptides for yeast host cells include, but are not limited to those fromthe genes for Saccharomyces cerevisiae alpha-factor and Saccharomycescerevisiae invertase. Useful signal peptides for mammalian host cellsinclude but are not limited to those from the genes for immunoglobulingamma (IgG).

In some embodiments, the control sequence is a propeptide coding regionthat codes for an amino acid sequence positioned at the amino terminusof a polypeptide. The resultant polypeptide is referred to as a“proenzyme,” “propolypeptide,” or “zymogen,” in some cases). Apropolypeptide can be converted to a mature active polypeptide bycatalytic or autocatalytic cleavage of the propeptide from thepropolypeptide.

In another aspect, the present invention also provides a recombinantexpression vector comprising a polynucleotide encoding an engineered GLApolypeptide, and one or more expression regulating regions such as apromoter and a terminator, a replication origin, etc., depending on thetype of hosts into which they are to be introduced. in some embodiments,the various nucleic acid and control sequences described above arejoined together to produce a recombinant expression vector whichincludes one or more convenient restriction sites to allow for insertionor substitution of the nucleic acid sequence encoding the variant GLApolypeptide at such sites. Alternatively, the polynucleotide sequence(s)of the present invention are expressed by inserting the polynucleotidesequence or a nucleic acid construct comprising the polynucleotidesequence into an appropriate vector for expression. In creating theexpression vector, the coding sequence is located in the vector so thatthe coding sequence is operably linked with the appropriate controlsequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus), that can be conveniently subjected to recombinant DNA proceduresand can result in the expression of the variant GLA polynucleotidesequence. The choice of the vector will typically depend on thecompatibility of the vector with the host cell into which the vector isto be introduced. The vectors may be linear or closed circular plasmids.

In some embodiments, the expression vector is an autonomouslyreplicating vector (i.e., a vector that exists as an extra-chromosomalentity, the replication of which is independent of chromosomalreplication, such as a plasmid, an extra-chromosomal element, aminichromosome, or an artificial chromosome). The vector may contain anymeans for assuring self-replication. In some alternative embodiments,the vector may be one which, when introduced into the host cell, isintegrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids which togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon may be used.

In some embodiments, the expression vector preferably contains one ormore selectable markers, which permit easy selection of transformedcells. A “selectable marker” is a gene the product of which provides forbiocide or viral resistance, resistance to heavy metals, prototrophy toauxotrophs, and the like. Suitable markers for yeast host cells include,but are not limited to ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3.Selectable markers for use in a filamentous fungal host cell include,but are not limited to, amdS (acetamidase), argB (ornithinecarbamoyltransferases), bar (phosphinothricin acetyltransferase), hph(hygromycin phosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),and trpC (anthranilate synthase), as well as equivalents thereof. Inanother aspect, the present invention provides a host cell comprising apolynucleotide encoding at least one engineered GLA polypeptide of thepresent application, the polynucleotide being operatively linked to oneor more control sequences for expression of the engineered GLA enzyme(s)in the host cell. Host cells for use in expressing the polypeptidesencoded by the expression vectors of the present invention are wellknown in the art and include but are not limited to, fungal cells, suchas yeast cells (e.g., Saccharomyces cerevisiae and Pichia pastoris[e.g., ATCC Accession No. 201178]); insect cells (e.g., Drosophila S2and Spodoptera Sf9 cells), plant cells, animal cells (e.g., CHO, COS,and BHK), and human cells (e.g., HEK293T, human fibroblast, THP-1,Jurkat and Bowes melanoma cell lines).

Accordingly, in another aspect, the present invention provides methodsfor producing the engineered GLA polypeptides, where the methodscomprise culturing a host cell capable of expressing a polynucleotideencoding the engineered GLA polypeptide under conditions suitable forexpression of the polypeptide. In some embodiments, the methods furthercomprise the steps of isolating and/or purifying the GLA polypeptides,as described herein.

Appropriate culture media and growth conditions for the above-describedhost cells are well known in the art. Polynucleotides for expression ofthe GLA polypeptides may be introduced into cells by various methodsknown in the art. Techniques include, among others, electroporation,biolistic particle bombardment, liposome mediated transfection, calciumchloride transfection, and protoplast fusion.

The engineered GLA with the properties disclosed herein can be obtainedby subjecting the polynucleotide encoding the naturally occurring orengineered GLA polypeptide to mutagenesis and/or directed evolutionmethods known in the art, and as described herein. An exemplary directedevolution technique is mutagenesis and/or DNA shuffling (See e.g.,Stemmer, Proc. Natl. Acad. Sci. USA 91:10747-10751 [1994]; WO 95/22625;WO 97/0078; WO 97/35966; WO 98/27230; WO 00/42651; WO 01/75767 and U.S.Pat. No. 6,537,746). Other directed evolution procedures that can beused include, among others, staggered extension process (StEP), in vitrorecombination (See e.g., Zhao et al., Nat. Biotechnol., 16:258-261[1998]), mutagenic PCR (See e.g., Caldwell et al., PCR Methods Appl.,3:S136-S140 [1994]), and cassette mutagenesis (See e.g., Black et al.,Proc. Natl. Acad. Sci. USA 93:3525-3529 [1996]).

For example, mutagenesis and directed evolution methods can be readilyapplied to polynucleotides to generate variant libraries that can beexpressed, screened, and assayed. Mutagenesis and directed evolutionmethods are well known in the art (See e.g., U.S. Pat. Nos. 5,605,793,5,811,238, 5,830,721, 5,834,252, 5,837,458, 5,928,905, 6,096,548,6,117,679, 6,132,970, 6,165,793, 6,180,406, 6,251,674, 6,277,638,6,287,861, 6,287,862, 6,291,242, 6,297,053, 6,303,344, 6,309,883,6,319,713, 6,319,714, 6,323,030, 6,326,204, 6,335,160, 6,335,198,6,344,356, 6,352,859, 6,355,484, 6,358,740, 6,358,742, 6,365,377,6,365,408, 6,368,861, 6,372,497, 6,376,246, 6,379,964, 6,387,702,6,391,552, 6,391,640, 6,395,547, 6,406,855, 6,406,910, 6,413,745,6,413,774, 6,420,175, 6,423,542, 6,426,224, 6,436,675, 6,444,468,6,455,253, 6,479,652, 6,482,647, 6,489,146, 6,506,602, 6,506,603,6,519,065, 6,521,453, 6,528,311, 6,537,746, 6,573,098, 6,576,467,6,579,678, 6,586,182, 6,602,986, 6,613,514, 6,653,072, 6,716,631,6,946,296, 6,961,664, 6,995,017, 7,024,312, 7,058,515, 7,105,297,7,148,054, 7,288,375, 7,421,347, 7,430,477, 7,534,564, 7,620,500,7,620,502, 7,629,170, 7,702,464, 7,747,391, 7,747,393, 7,751,986,7,776,598, 7,783,428, 7,795,030, 7,853,410, 7,868,138, 7,873,499,7,904,249, 7,957,912, 8,383,346, 8,504,498, 8,849,575, 8,876,066,8,768,871, and all related non-US counterparts; Ling et al., Anal.Biochem., 254(2):157-78 [1997]; Dale et al., Meth. Mol. Biol., 57:369-74[1996]; Smith, Ann. Rev. Genet., 19:423-462 [1985]; Botstein et al.,Science, 229:1193-1201 [1985]; Carter, Biochem. J., 237:1-7 [1986];Kramer et al., Cell, 38:879-887 [1984]; Wells et al., Gene, 34:315-323[1985]; Minshull et al., Curr. Op. Chem. Biol., 3:284-290 [1999];Christians et al., Nat. Biotechnol., 17:259-264 [1999]; Crameri et al.,Nature, 391:288-291 [1998]; Crameri, et al., Nat. Biotechnol.,15:436-438 [1997]; Zhang et al., Proc. Nat. Acad. Sci. U.S.A.,94:4504-4509 [1997]; Crameri et al., Nat. Biotechnol., 14:315-319[1996]; Stemmer, Nature, 370:389-391 [1994]; Stemmer, Proc. Nat. Acad.Sci. USA, 91:10747-10751 [1994]; US Pat. Appln. Publn. Nos.2008/0220990, US 2009/0312196, US2014/0005057, US2014/0214391,US2014/0221216; US2015/0050658, US2015/0133307, US2015/0134315 and allrelated non-US counterparts; WO 95/22625, WO 97/0078, WO 97/35966, WO98/27230, WO 00/42651, WO 01/75767, and WO 2009/152336; all of which areincorporated herein by reference).

In some embodiments, the enzyme variants obtained following mutagenesistreatment are screened by subjecting the enzyme variants to a definedtemperature (or other assay conditions) and measuring the amount ofenzyme activity remaining after heat treatments or other assayconditions. DNA containing the polynucleotide encoding the GLApolypeptide is then isolated from the host cell, sequenced to identifythe nucleotide sequence changes (if any), and used to express the enzymein a different or the same host cell. Measuring enzyme activity from theexpression libraries can be performed using any suitable method known inthe art (e.g., standard biochemistry techniques, such as HPLC analysis).

For engineered polypeptides of known sequence, the polynucleotidesencoding the enzyme can be prepared by standard solid-phase methods,according to known synthetic methods. In some embodiments, fragments ofup to about 100 bases can be individually synthesized, then joined(e.g., by enzymatic or chemical litigation methods, or polymerasemediated methods) to form any desired continuous sequence. For example,polynucleotides and oligonucleotides disclosed herein can be prepared bychemical synthesis using the classical phosphoramidite method (See e.g.,Beaucage et al., Tetra. Lett., 22:1859-69 [1981]; and Matthes et al.,EMBO J., 3:801-05 [1984]), as it is typically practiced in automatedsynthetic methods. According to the phosphoramidite method,oligonucleotides are synthesized (e.g., in an automatic DNAsynthesizer), purified, annealed, ligated and cloned in appropriatevectors.

Accordingly, in some embodiments, a method for preparing the engineeredGLA polypeptide can comprise: (a) synthesizing a polynucleotide encodinga polypeptide comprising an amino acid sequence selected from the aminoacid sequence of any variant provided in Table 2.1, 2.2, 2.3, 2.4, 2.5,and/or 6.1, as well as SEQ ID NOS:10, 13, 15, 18, 21, and/or 24, and (b)expressing the GLA polypeptide encoded by the polynucleotide. In someembodiments of the method, the amino acid sequence encoded by thepolynucleotide can optionally have one or several (e.g., up to 3, 4, 5,or up to 10) amino acid residue deletions, insertions and/orsubstitutions. In some embodiments, the amino acid sequence hasoptionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20,1-21, 1-22, 1-23, 1-24, 1-25, 1-30, 1-35, 1-40, 1-45, or 1-50 amino acidresidue deletions, insertions and/or substitutions. In some embodiments,the amino acid sequence has optionally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 30, 35,40, 45, or 50 amino acid residue deletions, insertions and/orsubstitutions. In some embodiments, the amino acid sequence hasoptionally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18,20, 21, 22, 23, 24, or 25 amino acid residue deletions, insertionsand/or substitutions. In some embodiments, the substitutions can beconservative or non-conservative substitutions.

The expressed engineered GLA polypeptide can be assessed for any desiredimproved property (e.g., activity, selectivity, stability, acidtolerance, protease sensitivity, etc.), using any suitable assay knownin the art, including but not limited to the assays and conditionsdescribed herein.

In some embodiments, any of the engineered GLA polypeptides expressed ina host cell are recovered from the cells and/or the culture medium usingany one or more of the well-known techniques for protein purification,including, among others, lysozyme treatment, sonication, filtration,salting-out, ultra-centrifugation, and chromatography.

Chromatographic techniques for isolation of the GLA polypeptidesinclude, among others, reverse phase chromatography high performanceliquid chromatography, ion exchange chromatography, hydrophobicinteraction chromatography, gel electrophoresis, and affinitychromatography. Conditions for purifying a particular enzyme depends, inpart, on factors such as net charge, hydrophobicity, hydrophilicity,molecular weight, molecular shape, etc., and will be apparent to thosehaving skill in the art. In some embodiments, affinity techniques may beused to isolate the improved variant GLA enzymes. In some embodimentsutilizing affinity chromatography purification, any antibody whichspecifically binds the variant GLA polypeptide finds use. In someembodiments utilizing affinity chromatography purification, proteinsthat bind to the glycans covalently attached to GLA find use. In stillother embodiments utilizing affinity-chromatography purifications, anysmall molecule that binds to the GLA active site finds use. For theproduction of antibodies, various host animals, including but notlimited to rabbits, mice, rats, etc., are immunized by injection with aGLA polypeptide (e.g., a GLA variant), or a fragment thereof. in someembodiments, the GLA polypeptide or fragment is attached to a suitablecarrier, such as BSA, by means of a side chain functional group orlinkers attached to a side chain functional group.

In some embodiments, the engineered GLA polypeptide is produced in ahost cell by a method comprising culturing a host cell (e.g., S.cerevisiae, Daucus carota, Nicotiana tabacum, H. sapiens (e.g.,HEK293T), or Cricetulus griseus (e.g., CHO)) comprising a polynucleotidesequence encoding an engineered GLA polypeptide as described hereinunder conditions conducive to the production of the engineered GLApolypeptide and recovering the engineered GLA polypeptide from the cellsand/or culture medium.

In some embodiments, the invention encompasses a method of producing anengineered GLA polypeptide comprising culturing a recombinant eukaryoticcell comprising a polynucleotide sequence encoding an engineered GLApolypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity toreference sequences SEQ ID NOS:5 and/or 10, and one or more amino acidresidue differences as compared to SEQ ID NO:5 and/or 10, selected fromthose provided in Tables 2.1, 2.2, 2.4, 2.5, and/or 6.1, and/orcombinations thereof when optimally aligned with the amino acid sequenceof SEQ ID NO:5 and/or 10, under suitable culture conditions to allow theproduction of the engineered GLA polypeptide and optionally recoveringthe engineered GLA polypeptide from the culture and/or culturedbacterial cells.

In some embodiments, once the engineered GLA polypeptides are recoveredfrom the recombinant host cells or cell culture medium, they are furtherpurified by any suitable method(s) known in the art. In some additionalembodiments, the purified GLA polypeptides are combined with otheringredients and compounds to provide compositions and formulationscomprising the engineered GLA polypeptide as appropriate for differentapplications and uses (e.g., pharmaceutical compositions). In someadditional embodiments, the purified GLA polypeptides, or the formulatedGLA polypeptides are lyophilized

Compositions:

The present invention provides various compositions and formats,including but not limited to those described below. In some embodiments,the present invention provides engineered GLA polypeptides suitable foruse in pharmaceutical and other compositions, such asdietary/nutritional supplements.

Depending on the mode of administration, these compositions comprising atherapeutically effective amount of an engineered GLA according to theinvention are in the form of a solid, semi-solid, or liquid. In someembodiments, the compositions include other pharmaceutically acceptablecomponents such as diluents, buffers, excipients, salts, emulsifiers,preservatives, stabilizers, fillers, and other ingredients. Details ontechniques for formulation and administration are well known in the artand described in the literature.

In some embodiments, the engineered GLA polypeptides are formulated foruse in pharmaceutical compositions. Any suitable format for use indelivering the engineered GLA polypeptides find use in the presentinvention, including but not limited to pills, tablets, gel tabs,capsules, lozenges, dragees, powders, soft gels, sol-gels, gels,emulsions, implants, patches, sprays, ointments, liniments, creams,pastes, jellies, paints, aerosols, chewing gums, demulcents, sticks,solutions, suspensions (including but not limited to oil-basedsuspensions, oil-in water emulsions, etc.), slurries, syrups, controlledrelease formulations, suppositories, etc. In some embodiments, theengineered GLA polypeptides are provided in a format suitable forinjection or infusion (i.e., in an injectable formulation). In someembodiments, the engineered GLA polypeptides are provided inbiocompatible matrices such as sol-gels, including silica-based (e.g.,oxysilane) sol-gels. In some embodiments, the engineered GLApolypeptides are encapsulated. In some alternative embodiments, theengineered GLA polypeptides are encapsulated in nanostructures (e.g.,nanotubes, nanotubules, nanocapsules, or microcapsules, microspheres,liposomes, etc.). Indeed, it is not intended that the present inventionbe limited to any particular delivery formulation and/or means ofdelivery. It is intended that the engineered GLA polypeptides beadministered by any suitable means known in the art, including but notlimited to parenteral, oral, topical, transdermal, intranasal,intraocular, intrathecal, via implants, etc.

In some embodiments, the engineered GLA polypeptides are chemicallymodified by glycosylation, chemical crosslinking reagents, pegylation(i.e., modified with polyethylene glycol [PEG] or activated PEG, etc.)or other compounds (See e.g., Ikeda, Amino Acids 29:283-287 [2005]; U.S.Pat. Nos. 7,531,341, 7,534,595, 7,560,263, and 7,53,653; US Pat. Appln.Publ. Nos. 2013/0039898, 2012/0177722, etc.). Indeed, it is not intendedthat the present invention be limited to any particular delivery methodand/or mechanism.

In some additional embodiments, the engineered GLA polypeptides areprovided in formulations comprising matrix-stabilized enzyme crystals.In some embodiments, the formulation comprises a cross-linkedcrystalline engineered GLA enzyme and a polymer with a reactive moietythat adheres to the enzyme crystals. The present invention also providesengineered GLA polypeptides in polymers.

In some embodiments, compositions comprising the engineered GLApolypeptides of the present invention include one or more commonly usedcarrier compounds, including but not limited to sugars (e.g., lactose,sucrose, mannitol, and/or sorbitol), starches (e.g., corn, wheat, rice,potato, or other plant starch), cellulose (e.g., methyl cellulose,hydroxypropylmethyl cellulose, sodium carboxy-methylcellulose), gums(e.g., arabic, tragacanth, guar, etc.), and/or proteins (e.g., gelatin,collagen, etc.).

In some embodiments, the present invention provides engineered GLApolypeptides suitable for use in decreasing the concentration ofglycolipids in fluids such as blood, cerebrospinal fluid, etc. Thedosage of engineered GLA polypeptide(s) administered depends upon thecondition or disease, the general condition of the subject, and otherfactors known to those in the art. In some embodiments, the compositionsare intended for single or multiple administrations. In someembodiments, it is contemplated that the concentration of engineered GLApolypeptide(s) in the composition(s) administered to a human with Fabrydisease is sufficient to effectively treat, and/or ameliorate disease(e.g., Fabry disease). In some embodiments, the engineered GLApolypeptides are administered in combination with other pharmaceuticaland/or dietary compositions.

EXPERIMENTAL

The following Examples, including experiments and results achieved, areprovided for illustrative purposes only and are not to be construed aslimiting the present invention.

In the experimental disclosure below, the following abbreviations apply:ppm (parts per million); M (molar); mM (millimolar), uM and μM(micromolar); nM (nanomolar); mol (moles); gm and g (gram); mg(milligrams); ug and μg (micrograms); L and 1 (liter); ml and mL(milliliter); cm (centimeters); mm (millimeters); um and μm(micrometers); sec. (seconds); min(s) (minute(s)); h(s) and hr(s)(hour(s)); U (units); MW (molecular weight); rpm (rotations per minute);° C. (degrees Centigrade); CDS (coding sequence); DNA (deoxyribonucleicacid); RNA (ribonucleic acid); E. coli W3110 (commonly used laboratoryE. coli strain, available from the Coli Genetic Stock Center [CGSC], NewHaven, Conn.); HPLC (high pressure liquid chromatography); MWCO(molecular weight cut-off); SDS-PAGE (sodium dodecyl sulfatepolyacrylamide gel electrophoresis); PES (polyethersulfone); CFSE(carboxyfluorescein succinimidyl ester); IPTG (isopropylβ-D-1-thiogalactopyranoside); PMBS (polymyxin B sulfate); NADPH(nicotinamide adenine dinucleotide phosphate); GIDH (glutamatedehydrogenase); FIOPC (fold improvements over positive control); PBMC(peripheral blood mononuclear cells); LB (Luria broth); MeOH (methanol);Athens Research (Athens Research Technology, Athens, Ga.); ProSpec(ProSpec Tany Technogene, East Brunswick, N.J.); Sigma-Aldrich(Sigma-Aldrich, St. Louis, Mo.); Ram Scientific (Ram Scientific, Inc.,Yonkers, N.Y.); Pall Corp. (Pall, Corp., Pt. Washington, N.Y.);Millipore (Millipore, Corp., Billerica Mass.); Difco (DifcoLaboratories, BD Diagnostic Systems, Detroit, Mich.); Molecular Devices(Molecular Devices, LLC, Sunnyvale, Calif.); Kuhner (Adolf Kuhner, AG,Basel, Switzerland); Axygen (Axygen, Inc., Union City, Calif.); TorontoResearch Chemicals (Toronto Research Chemicals Inc., Toronto, Ontario,Canada); Cambridge Isotope Laboratories, (Cambridge IsotopeLaboratories, Inc., Tewksbury, Mass.); Applied Biosystems (AppliedBiosystems, part of Life Technologies, Corp., Grand Island, N.Y.),Agilent (Agilent Technologies, Inc., Santa Clara, Calif.); ThermoScientific (part of Thermo Fisher Scientific, Waltham, Mass.); Corning(Corning, Inc., Palo Alto, Calif.); Megazyme (Megazyme International,Wicklow, Ireland); Enzo (Enzo Life Sciences, Inc., Farmingdale, N.Y.);GE Healthcare (GE Healthcare Bio-Sciences, Piscataway, N.J.); Pierce(Pierce Biotechnology (now part of Thermo Fisher Scientific), Rockford,Ill.); LI-COR (LI-COR Biotechnology, Lincoln, Nebr.); Amicus (AmicusTherapeutics, Cranbury, N.J.); Phenomenex (Phenomenex, Inc., Torrance,Calif.); Optimal (Optimal Biotech Group, Belmont, Calif.); and Bio-Rad(Bio-Rad Laboratories, Hercules, Calif.).

The following polynucleotide and polypeptide sequences find use in thepresent invention. In some cases (as shown below), the polynucleotidesequence is followed by the encoded polypeptide.

Polynucleotide sequence of full length human GLA cDNA (SEQ ID NO. 1):(SEQ ID NO: 1) ATGCAGCTGAGGAACCCAGAACTACATCTGGGCTGCGCGCTTGCGCTTCGCTTCCTGGCCCTCGTTTCCTGGGACATCCCTGGGGCTAGAGCACTGGACAATGGATTGGCAAGGACGCCTACCATGGGCTGGCTGCACTGGGAGCGCTTCATGTGCAACCTTGACTGCCAGGAAGAGCCAGATTCCTGCATCAGTGAGAAGCTCTTCATGGAGATGGCAGAGCTCATGGTCTCAGAAGGCTGGAAGGATGCAGGTTATGAGTACCTCTGCATTGATGACTGTTGGATGGCTCCCCAAAGAGATTCAGAAGGCAGACTTCAGGCAGACCCTCAGCGCTTTCCTCATGGGATTCGCCAGCTAGCTAATTATGTTCACAGCAAAGGACTGAAGCTAGGGATTTATGCAGATGTTGGAAATAAAACCTGCGCAGGCTTCCCTGGGAGTTTTGGATACTACGACATTGATGCCCAGACCTTTGCTGACTGGGGAGTAGATCTGCTAAAATTTGATGGTTGTTACTGTGACAGTTTGGAAAATTTGGCAGATGGTTATAAGCACATGTCCTTGGCCCTGAATAGGACTGGCAGAAGCATTGTGTACTCCTGTGAGTGGCCTCTTTATATGTGGCCCTTTCAAAAGCCCAATTATACAGAAATCCGACAGTACTGCAATCACTGGCGAAATTTTGCTGACATTGATGATTCCTGGAAAAGTATAAAGAGTATCTTGGACTGGACATCTTTTAACCAGGAGAGAATTGTTGATGTTGCTGGACCAGGGGGTTGGAATGACCCAGATATGTTAGTGATTGGCAACTTTGGCCTCAGCTGGAATCAGCAAGTAACTCAGATGGCCCTCTGGGCTATCATGGCTGCTCCTTTATTCATGTCTAATGACCTCCGACACATCAGCCCTCAAGCCAAAGCTCTCCTTCAGGATAAGGACGTAATTGCCATCAATCAGGACCCCTTGGGCAAGCAAGGGTACCAGCTTAGACAGGGAGACAACTTTGAAGTGTGGGAACGACCTCTCTCAGGCTTAGCCTGGGCTGTAGCTATGATAAACCGGCAGGAGATTGGTGGACCTCGCTCTTATACCATCGCAGTTGCTTCCCTGGGTAAAGGAGTGGCCTGTAATCCTGCCTGCTTCATCACACAGCTCCTCCCTGTGAAAAGGAAGCTAGGGTTCTATGAATGGACTTCAAGGTTAAGAAGTCACATAAATCCCACAGGCACTGTTTTGCTTCAGCTAGAAAATACAATGCAGATGTCATTAAAAGACTTACTTTAG Polypeptide sequence of full lengthhuman GLA: (SEQ ID NO: 2) MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCNLDCQEEPDSCISEKLFMEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGNKTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKHMSLALNRTGRSIVYSCEWPLYMWPFQKPNYTEIRQYCNHWRNFADIDDSWKSIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFGLSWNQQVTQMALWAIMAAPLFMSNDLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDNFEVWERPLSGLAWAVAMINRQEIGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSHINPTGTVLLQLE NTMQMSLKDLLPolynucleotide sequence of mature yeastcodon-optimized (yCDS) human GLA: (SEQ ID NO: 3)TTGGATAACGGGTTAGCCCGTACACCTACTATGGGTTGGCTTCACTGGGAAAGATTCATGTGTAACTTAGATTGCCAAGAAGAGCCTGACAGCTGTATCTCAGAGAAACTATTCATGGAGATGGCTGAACTAATGGTAAGTGAAGGATGGAAGGATGCTGGTTATGAATACCTATGTATTGATGATTGCTGGATGGCTCCACAGCGTGATTCAGAAGGTAGGTTACAAGCTGACCCCCAGAGATTCCCACATGGCATACGTCAGCTTGCAAACTACGTACACAGCAAGGGTCTAAAGTTAGGCATCTACGCTGATGTCGGAAACAAGACATGTGCTGGTTTCCCAGGTTCATTCGGTTACTATGACATAGATGCGCAGACGTTTGCTGATTGGGGTGTTGATTTGTTGAAGTTTGATGGATGCTACTGCGATTCCCTGGAGAACCTAGCCGATGGGTACAAACACATGAGTTTGGCTCTAAACAGGACTGGTAGGAGCATCGTCTATAGTTGTGAATGGCCCTTGTACATGTGGCCGTTTCAGAAGCCAAACTACACTGAGATAAGACAATACTGTAACCATTGGCGTAACTTTGCTGACATAGATGATTCATGGAAGTCAATCAAATCTATCTTGGATTGGACTTCTTTCAACCAGGAAAGAATTGTTGATGTTGCAGGTCCAGGTGGATGGAATGACCCTGATATGCTTGTCATAGGGAACTTTGGGCTATCATGGAATCAACAAGTTACACAAATGGCTTTGTGGGCGATCATGGCCGCACCCCTATTCATGTCTAATGATCTACGTCACATATCACCCCAAGCAAAGGCTTTACTTCAAGATAAGGATGTCATAGCGATCAACCAAGATCCTCTTGGTAAACAAGGTTATCAATTGAGACAAGGTGACAACTTTGAAGTGTGGGAAAGACCATTGTCTGGACTTGCGTGGGCTGTTGCTATGATCAACCGTCAAGAGATCGGAGGGCCAAGATCTTACACTATCGCGGTAGCCTCTTTGGGTAAGGGTGTTGCGTGCAATCCTGCCTGCTTCATTACACAATTGCTTCCAGTTAAGAGAAAGTTGGGTTTCTATGAGTGGACATCTAGGCTAAGAAGTCACATCAATCCTACTGGTACGGTATTGTTGCAATTGGAGAACACAATGCAAATGTC TTTGAAAGATTTGTTAPolynucleotide sequence of mature human GLA (native hCDS):(SEQ ID NO: 4) CTGGACAATGGATTGGCAAGGACGCCTACCATGGGCTGGCTGCACTGGGAGCGCTTCATGTGCAACCTTGACTGCCAGGAAGAGCCAGATTCCTGCATCAGTGAGAAGCTCTTCATGGAGATGGCAGAGCTCATGGTCTCAGAAGGCTGGAAGGATGCAGGTTATGAGTACCTCTGCATTGATGACTGTTGGATGGCTCCCCAAAGAGATTCAGAAGGCAGACTTCAGGCAGACCCTCAGCGCTTTCCTCATGGGATTCGCCAGCTAGCTAATTATGTTCACAGCAAAGGACTGAAGCTAGGGATTTATGCAGATGTTGGAAATAAAACCTGCGCAGGCTTCCCTGGGAGTTTTGGATACTACGACATTGATGCCCAGACCTTTGCTGACTGGGGAGTAGATCTGCTAAAATTTGATGGTTGTTACTGTGACAGTTTGGAAAATTTGGCAGATGGTTATAAGCACATGTCCTTGGCCCTGAATAGGACTGGCAGAAGCATTGTGTACTCCTGTGAGTGGCCTCTTTATATGTGGCCCTTTCAAAAGCCCAATTATACAGAAATCCGACAGTACTGCAATCACTGGCGAAATTTTGCTGACATTGATGATTCCTGGAAAAGTATAAAGAGTATCTTGGACTGGACATCTTTTAACCAGGAGAGAATTGTTGATGTTGCTGGACCAGGGGGTTGGAATGACCCAGATATGTTAGTGATTGGCAACTTTGGCCTCAGCTGGAATCAGCAAGTAACTCAGATGGCCCTCTGGGCTATCATGGCTGCTCCTTTATTCATGTCTAATGACCTCCGACACATCAGCCCTCAAGCCAAAGCTCTCCTTCAGGATAAGGACGTAATTGCCATCAATCAGGACCCCTTGGGCAAGCAAGGGTACCAGCTTAGACAGGGAGACAACTTTGAAGTGTGGGAACGACCTCTCTCAGGCTTAGCCTGGGCTGTAGCTATGATAAACCGGCAGGAGATTGGTGGACCTCGCTCTTATACCATCGCAGTTGCTTCCCTGGGTAAAGGAGTGGCCTGTAATCCTGCCTGCTTCATCACACAGCTCCTCCCTGTGAAAAGGAAGCTAGGGTTCTATGAATGGACTTCAAGGTTAAGAAGTCACATAAATCCCACAGGCACTGTTTTGCTTCAGCTAGAAAATACAATGCAGATGTC ATTAAAAGACTTACTTPolypeptide sequence of mature Human GLA (SEQ ID NO. 5): (SEQ ID NO: 5)LDNGLARTPTMGWLHWERFMCNLDCQEEPDSCISEKLFMEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGNKTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKHMSLALNRTGRSIVYSCEWPLYMWPFQKPNYTEIRQYCNHWRNFADIDDSWKSIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFGLSWNQQVTQMALWAIMAAPLFMSNDLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDNFEVWERPLSGLAWAVAMINRQEIGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSHINPTG TVLLQLENTMQMSLKDLLPolynucleotide sequence of pCK110900i E. coli expression vector:(SEQ ID NO: 6) TCGAGTTAATTAAGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACGGCTATGACCATGATTACGGATTCACTGGCCGTCGTTTTACAATCTAGAGGCCAGCCTGGCCATAAGGAGATATACATATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTCTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAGCGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGTTTTTTTGCACACCATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTACAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGTAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGGGCCAAACTGGCCACCATCACCATCACCATTAGGGAAGAGCAGATGGGCAAGCTTGACCTGTGAAGTGAAAAATGGCGCACATTGTGCGACATTTTTTTTTGAATTCTACGTAAAAAGCCGCCGATACATCGGCTGCTTTTTTTTTGATAGAGGTTCAAACTTGTGGTATAATGAAATAAGATCACTCCGGGGCGTATTTTTTGAGTTATCGAGATTTTCAGGAGCTAAGGAAGCTAAAATGGAGAAAAAAATCACTGGATATACCACCGTTGATATATCCCAATGGCATCGTAAAGAACATTTTGAGGCATTTCAGTCAGTTGCTCAATGTACCTATAACCAGACCGTTCAGCTGGATATTACGGCCTTTTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGCCTTTATTCACATTCTTGCCCGCCTGATGAATGCTCATCCGGAGTTCCGTATGGCAATGAAAGACGGTGAGCTGGTGATATGGGATAGTGTTCACCCTTGTTACACCGTTTTCCATGAGCAAACTGAAACGTTTTCATCGCTCTGGAGTGAATACCACGACGATTTCCGGCAGTTTCTACACATATATTCGCAAGATGTGGCGTGTTACGGTGAAAACCTGGCCTATTTCCCTAAAGGGTTTATTGAGAATATGTTTTTCGTCTCAGCCAATCCCTGGGTGAGTTTCACCAGTTTTGATTTAAACGTGGCCAATATGGACAACTTCTTCGCCCCCGTTTTCACCATGGGCAAATATTATACGCAAGGCGACAAGGTGCTGATGCCGCTGGCGATTCAGGTTCATCATGCCGTCTGTGATGGCTTCCATGTCGGCAGAATGCTTAATGAATTACAACAGTACTGCGATGAGTGGCAGGGCGGGGCGTAACTGCAGGAGCTCAAACAGCAGCCTGTATTCAGGCTGCTTTTTTCGTTTTGGTCTGCGCGTAATCTCTTGCTCTGAAAACGAAAAAACCGCCTTGCAGGGCGGTTTTTCGAAGGTTCTCTGAGCTACCAACTCTTTGAACCGAGGTAACTGGCTTGGAGGAGCGCAGTCACCAAAACTTGTCCTTTCAGTTTAGCCTTAACCGGCGCATGACTTCAAGACTAACTCCTCTAAATCAATTACCAGTGGCTGCTGCCAGTGGTGCTTTTGCATGTCTTTCCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGACTGAACGGGGGGTTCGTGCATACAGTCCAGCTTGGAGCGAACTGCCTACCCGGAACTGAGTGTCAGGCGTGGAATGAGACAAACGCGGCCATAACAGCGGAATGACACCGGTAAACCGAAAGGCAGGAACAGGAGAGCGCACGAGGGAGCCGCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCACTGATTTGAGCGTCAGATTTCGTGATGCTTGTCAGGGGGGCGGAGCCTATGGAAAAACGGCTTTGCCGCGGCCCTCTCACTTCCCTGTTAAGTATCTTCCTGGCATCTTCCAGGAAATCTCCGCCCCGTTCGTAAGCCATTTCCGCTCGCCGCAGTCGAACGACCGAGCGTAGCGAGTCAGTGAGCGAGGAAGCGGAATATATCCTGTATCACATATTCTGCTGACGCACCGGTGCAGCCTTTTTTCTCCTGCCACATGAAGCACTTCACTGACACCCTCATCAGTGAACCACCGCTGGTAGCGGTGGTTTTTTTAGGCCTATGGCCTTTTTTTTTTGTGGGAAACCTTTCGCGGTATGGTATTAAAGCGCCCGGAAGAGAGTCAATTCAGGGTGGTGAATGTGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATCGTTTCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGACATCTCGGTAGTGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAGGGCCAGGCGGTTAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGGTACCCGATAAAAGCGGCTTCCTGACAGGAGGCCGTTTTG TTTCPolynucleotide sequence of pYT-72Bgl secreted yeast expression vector:(SEQ ID NO: 7) TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGTACAAATATCATAAAAAAAGAGAATCTTTTTAAGCAAGGATTTTCTTAACTTCTTCGGCGACAGCATCACCGACTTCGGTGGTACTGTTGGAACCACCTAAATCACCAGTTCTGATACCTGCATCCAAAACCTTTTTAACTGCATCTTCAATGGCTTTACCTTCTTCAGGCAAGTTCAATGACAATTTCAACATCATTGCAGCAGACAAGATAGTGGCGATAGGGTTGACCTTATTCTTTGGCAAATCTGGAGCGGAACCATGGCATGGTTCGTACAAACCAAATGCGGTGTTCTTGTCTGGCAAAGAGGCCAAGGACGCAGATGGCAACAAACCCAAGGAGCCTGGGATAACGGAGGCTTCATCGGAGATGATATCACCAAACATGTTGCTGGTGATTATAATACCATTTAGGTGGGTTGGGTTCTTAACTAGGATCATGGCGGCAGAATCAATCAATTGATGTTGAACTTTCAATGTAGGGAATTCGTTCTTGATGGTTTCCTCCACAGTTTTTCTCCATAATCTTGAAGAGGCCAAAACATTAGCTTTATCCAAGGACCAAATAGGCAATGGTGGCTCATGTTGTAGGGCCATGAAAGCGGCCATTCTTGTGATTCTTTGCACTTCTGGAACGGTGTATTGTTCACTATCCCAAGCGACACCATCACCATCGTCTTCCTTTCTCTTACCAAAGTAAATACCTCCCACTAATTCTCTAACAACAACGAAGTCAGTACCTTTAGCAAATTGTGGCTTGATTGGAGATAAGTCTAAAAGAGAGTCGGATGCAAAGTTACATGGTCTTAAGTTGGCGTACAATTGAAGTTCTTTACGGATTTTTAGTAAACCTTGTTCAGGTCTAACACTACCGGTACCCCATTTAGGACCACCCACAGCACCTAACAAAACGGCATCAGCCTTTTTGGAGGCTTCCAGCGCCTCATTTGGAAGTGGAACACCTGTAGCATCGATAGCAGCCCCCCCAATTAAATGATTTTCGAAATCGAACTTGACATTGGAACGAACATCAGAAATAGCTTTAAGAACCTTAATGGCTTCGGCTGTGATTTCTTGACCAACGTGGTCACCTGGCAAAACGACGATTTTTTTAGGGGCAGACATTACAATGGTATATCCTTGAAATATATATAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATGCAGCTTCTCAATGATATTCGAATACGCTTTGAGGAGATACAGCCTAATATCCGACAAACTGTTTTACAGATTTACGATCGTACTTGTTACCCATCATTGAATTTTGAACATCCGAACCTGGGAGTTTTCCCTGAAACAGATAGTATATTTGAACCTGTATAATAATATATAGTCTAGCGCTTTACGGAAGACAATGTATGTATTTCGGTTCCTGGAGAAACTATTGCATCTATTGCATAGGTAATCTTGCACGTCGCATCCCCGGTTCATTTTCTGCGTTTCCATCTTGCACTTCAATAGCATATCTTTGTTAACGAAGCATCTGTGCTTCATTTTGTAGAACAAAAATGCAACGCGAGAGCGCTAATTTTTCAAACAAAGAATCTGAGCTGCATTTTTACAGAACAGAAATGCAACGCGAAAGCGCTATTTTACCAACGAAGAATCTGTGCTTCATTTTTGTAAAACAAAAATGCAACGCGAGAGCGCTAATTTTTCAAACAAAGAATCTGAGCTGCATTTTTACAGAACAGAAATGCAACGCGAGAGCGCTATTTTACCAACAAAGAATCTATACTTCTTTTTTGTTCTACAAAAATGCATCCCGAGAGCGCTATTTTTCTAACAAAGCATCTTAGATTACTTTTTTTCTCCTTTGTGCGCTCTATAATGCAGTCTCTTGATAACTTTTTGCACTGTAGGTCCGTTAAGGTTAGAAGAAGGCTACTTTGGTGTCTATTTTCTCTTCCATAAAAAAAGCCTGACTCCACTTCCCGCGTTTACTGATTACTAGCGAAGCTGCGGGTGCATTTTTTCAAGATAAAGGCATCCCCGATTATATTCTATACCGATGTGGATTGCGCATACTTTGTGAACAGAAAGTGATAGCGTTGATGATTCTTCATTGGTCAGAAAATTATGAACGGTTTCTTCTATTTTGTCTCTATATACTACGTATAGGAAATGTTTACATTTTCGTATTGTTTTCGATTCACTCTATGAATAGTTCTTACTACAATTTTTTTGTCTAAAGAGTAATACTAGAGATAAACATAAAAAATGTAGAGGTCGAGTTTAGATGCAAGTTCAAGGAGCGAAAGGTGGATGGGTAGGTTATATAGGGATATAGCACAGAGATATATAGCAAAGAGATACTTTTGAGCAATGTTTGTGGAAGCGGTATTCGCAATATTTTAGTAGCTCGTTACAGTCCGGTGCGTTTTTGGTTTTTTGAAAGTGCGTCTTCAGAGCGCTTTTGGTTTTCAAAAGCGCTCTGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGGAATAGGAACTTCAAAGCGTTTCCGAAAACGAGCGCTTCCGAAAATGCAACGCGAGCTGCGCACATACAGCTCACTGTTCACGTCGCACCTATATCTGCGTGTTGCCTGTATATATATATACATGAGAAGAACGGCATAGTGCGTGTTTATGCTTAAATGCGTACTTATATGCGTCTATTTATGTAGGATGAAAGGTAGTCTAGTACCTCCTGTGATATTATCCCATTCCATGCGGGGTATCGTATGCTTCCTTCAGCACTACCCTTTAGCTGTTCTATATGCTGCCACTCCTCAATTGGATTAGTCTCATCCTTCAATGCTATCATTTCCTTTGATATTGGATCATATGCATAGTACCGAGAAACTAGTGCGAAGTAGTGATCAGGTATTGCTGTTATCTGATGAGTATACGTTGTCCTGGCCACGGCAGAAGCACGCTTATCGCTCCAATTTCCCACAACATTAGTCAACTCCGTTAGGCCCTTCATTGAAAGAAATGAGGTCATCAAATGTCTTCCAATGTGAGATTTTGGGCCATTTTTTATAGCAAAGATTGAATAAGGCGCATTTTTCTTCAAAGCTTTATTGTACGATCTGACTAAGTTATCTTTTAATAATTGGTATTCCTGTTTATTGCTTGAAGAATTGCCGGTCCTATTTACTCGTTTTAGGACTGGTTCAGAATTCCTCAAAAATTCATCCAAATATACAAGTGGATCGATGATAAGCTGTCAAACATGAGAATTCTTGAAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAACATGCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACGATCATGCGCACCCGTGGCCAGGACCCAACGCTGCCCGAGATGCGCCGCGTGCGGCTGCTGGAGATGGCGGACGCGATGGATATGTTCTGCCAAGGGTTGGTTTGCGCATTCACAGTTCTCCGCAAGAATTGATTGGCTCCAATTCTTGGAGTGGTGAATCCGTTAGCGAGGTGCCGCCGGCTTCCATTCAGGTCGAGGTGGCCCGGCTCCATGCACCGCGACGCAACGCGGGGAGGCAGACAAGGTATAGGGCGGCGCCTACAATCCATGCCAACCCGTTCCATGTGCTCGCCGAGGCGGCATAAATCGCCGTGACGATCAGCGGTCCAATGATCGAAGTTAGGCTGGTAAGAGCCGCGAGCGATCCTTGAAGCTGTCCCTGATGGTCGTCATCTACCTGCCTGGACAGCATGGCCTGCAACGCGGGCATCCCGATGCCGCCGGAAGCGAGAAGAATCATAATGGGGAAGGCCATCCAGCCTCGCGTCGCGAACGCCAGCAAGACGTAGCCCAGCGCGTCGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGGATCTGGGCAAAACGTAGGGGCAAACAAACGGAAAAATCGTTTCTCAAATTTTCTGATGCCAAGAACTCTAACCAGTCTTATCTAAAAATTGCCTTATGATCCGTCTCTCCGGTTACAGCCTGTGTAACTGATTAATCCTGCCTTTCTAATCACCATTCTAATGTTTTAATTAAGGGATTTTGTCTTCATTAACGGCTTTCGCTCATAAAAATGTTATGACGTTTTGCCCGCAGGCGGGAAACCATCCACTTCACGAGACTGATCTCCTCTGCCGGAACACCGGGCATCTCCAACTTATAAGTTGGAGAAATAAGAGAATTTCAGATTGAGAGAATGAAAAAAAAAAAAAAAAAAAGGCAGAGGAGAGCATAGAAATGGGGTTCACTTTTTGGTAAAGCTATAGCATGCCTATCACATATAAATAGAGTGCCAGTAGCGACTTTTTTCACACTCGAAATACTCTTACTACTGCTCTCTTGTTGTTTTTATCACTTCTTGTTTCTTCTTGGTAAATAGAATATCAAGCTACAAAAAGCATACAATCAACTATCAACTATTAACTATATCGTAATACACAGGATCCACCATGAAGGCTGCTGCGCTTTCCTGCCTCTTCGGCAGTACCCTTGCCGTTGCAGGCGCCATTGAATCGAGAAAGGTTCACCAGAAGCCCCTCGCGAGATCTGAACCTTTTTACCCGTCGCCATGGATGAATCCCAACGCCATCGGCTGGGCGGAGGCCTATGCCCAGGCCAAGTCCTTTGTCTCCCAAATGACTCTGCTAGAGAAGGTCAACTTGACCACGGGAGTCGGCTGGGGGGAGGAGCAGTGCGTCGGCAACGTGGGCGCGATCCCTCGCCTTGGACTTCGCAGTCTGTGCATGCATGACTCCCCTCTCGGCGTGCGAGGAACCGACTACAACTCAGCGTTCCCCTCTGGCCAGACCGTTGCTGCTACCTGGGATCGCGGTCTGATGTACCGTCGCGGCTACGCAATGGGCCAGGAGGCCAAAGGCAAGGGCATCAATGTCCTTCTCGGACCAGTCGCCGGCCCCCTTGGCCGCATGCCCGAGGGCGGTCGTAACTGGGAAGGCTTCGCTCCGGATCCCGTCCTTACCGGCATCGGCATGTCCGAGACGATCAAGGGCATTCAGGATGCTGGCGTCATCGCTTGTGCGAAGCACTTTATTGGAAACGAGCAGGAGCACTTCAGACAGGTGCCAGAAGCCCAGGGATACGGTTACAACATCAGCGAAACCCTCTCCTCCAACATTGACGACAAGACCATGCACGAGCTCTACCTTTGGCCGTTTGCCGATGCCGTCCGGGCCGGCGTCGGCTCTGTCATGTGCTCGTACAACCAGGGCAACAACTCGTACGCCTGCCAGAACTCGAAGCTGCTGAACGACCTCCTCAAGAACGAGCTTGGGTTTCAGGGCTTCGTCATGAGCGACTGGTGGGCACAGCACACTGGCGCAGCAAGCGCCGTGGCTGGTCTCGATATGTCCATGCCGGGCGACACCATGGTCAACACTGGCGTCAGTTTCTGGGGCGCCAATCTCACCCTCGCCGTCCTCAACGGCACAGTCCCTGCCTACCGTCTCGACGACATGTGCATGCGCATCATGGCCGCCCTCTTCAAGGTCACCAAGACCACCGACCTGGAACCGATCAACTTCTCCTTCTGGACCCGCGACACTTATGGCCCGATCCACTGGGCCGCCAAGCAGGGCTACCAGGAGATTAATTCCCACGTTGACGTCCGCGCCGACCACGGCAACCTCATCCGGAACATTGCCGCCAAGGGTACGGTGCTGCTGAAGAATACCGGCTCTCTACCCCTGAACAAGCCAAAGTTCGTGGCCGTCATCGGCGAGGATGCTGGGCCGAGCCCCAACGGGCCCAACGGCTGCAGCGACCGCGGCTGTAACGAAGGCACGCTCGCCATGGGCTGGGGATCCGGCACAGCCAACTATCCGTACCTCGTTTCCCCCGACGCCGCGCTCCAGGCGCGGGCCATCCAGGACGGCACGAGGTACGAGAGCGTCCTGTCCAACTACGCCGAGGAAAATACAAAGGCTCTGGTCTCGCAGGCCAATGCAACCGCCATCGTCTTCGTCAATGCCGACTCAGGCGAGGGCTACATCAACGTGGACGGTAACGAGGGCGACCGTAAGAACCTGACTCTCTGGAACAACGGTGATACTCTGGTCAAGAACGTCTCGAGCTGGTGCAGCAACACCATCGTCGTCATCCACTCGGTCGGCCCGGTCCTCCTGACCGATTGGTACGACAACCCCAACATCACGGCCATTCTCTGGGCTGGTCTTCCGGGCCAGGAGTCGGGCAACTCCATCACCGACGTGCTTTACGGCAAGGTCAACCCCGCCGCCCGCTCGCCCTTCACTTGGGGCAAGACCCGCGAAAGCTATGGCGCGGACGTCCTGTACAAGCCGAATAATGGCAATTGGGCGCCCCAACAGGACTTCACCGAGGGCGTCTTCATCGACTACCGCTACTTCGACAAGGTTGACGATGACTCGGTCATCTACGAGTTCGGCCACGGCCTGAGCTACACCACCTTCGAGTACAGCAACATCCGCGTCGTCAAGTCCAACGTCAGCGAGTACCGGCCCACGACGGGCACCACGATTCAGGCCCCGACGTTTGGCAACTTCTCCACCGACCTCGAGGACTATCTCTTCCCCAAGGACGAGTTCCCCTACATCCCGCAGTACATCTACCCGTACCTCAACACGACCGACCCCCGGAGGGCCTCGGGCGATCCCCACTACGGCCAGACCGCCGAGGAGTTCCTCCCGCCCCACGCCACCGATGACGACCCCCAGCCGCTCCTCCGGTCCTCGGGCGGAAACTCCCCCGGCGGCAACCGCCAGCTGTACGACATTGTCTACACAATCACGGCCGACATCACGAATACGGGCTCCGTTGTAGGCGAGGAGGTACCGCAGCTCTACGTCTCGCTGGGCGGTCCCGAGGATCCCAAGGTGCAGCTGCGCGACTTTGACAGGATGCGGATCGAACCCGGCGAGACGAGGCAGTTCACCGGCCGCCTGACGCGCAGAGATCTGAGCAACTGGGACGTCACGGTGCAGGACTGGGTCATCAGCAGGTATCCCAAGACGGCATATGTTGGGAGGAGCAGCCGGAAGTTGGATCTCAAGATTGAGCTTCCTTGATAAGTCGACCTCGACTTTGTTCCCACTGTACTTTTAGCTCGTACAAAATACAATATACTTTTCATTTCTCCGTAAACAACATGTTTTCCCATGTAATATCCTTTTCTATTTTTCGTTCCGTTACCAACTTTACACATACTTTATATAGCTATTCACTTCTATACACTAAAAAACTAAGACAATTTTAATTTTGCTGCCTGCCATATTTCAATTTGTTATAAATTCCTATAATTTATCCTATTAGTAGCTAAAAAAAGATGAATGTGAATCGAATCCTAAGAGAATTGGATCTGATCCACAGGACGGGTGTGGTCGCCATGATCGCGTAGTCGATAGTGGCTCCAAGTAGCGAAGCGAGCAGGACTGGGCGGCGGCCAAAGCGGTCGGACAGTGCTCCGAGAACGGGTGCGCATAGAAATTGCATCAACGCATATAGCGCTAGCAGCACGCCATAGTGACTGGCGATGCTGTCGGAATGGACGATATCCCGCAAGAGGCCCGGCAGTACCGGCATAACCAAGCCTATGCCTACAGCATCCAGGGTGACGGTGCCGAGGATGACGATGAGCGCATTGTTAGATTTCATACACGGTGCCTGACTGCGTTAGCAATTTAACTGTGATAAACTACCGCATTAAAGCTTTTTCTTTCCAATTTTTTTTTTTTCGTCATTATAAAAATCATTACGACCGAGATTCCCGGGTAATAACTGATATAATTAAATTGAAGCTCTAATTTGTGAGTTTAGTATACATGCATTTACTTATAATACAGTTTTTTAGTTTTGCTGGCCGCATCTTCTCAAATATGCTTCCCAGCCTGCTTTTCTGTAACGTTCACCCTCTACCTTAGCATCCCTTCCCTTTGCAAATAGTCCTCTTCCAACAATAATAATGTCAGATCCTGTAGAGACCACATCATCCACGGTTCTATACTGTTGACCCAATGCGTCTCCCTTGTCATCTAAACCCACACCGGGTGTCATAATCAACCAATCGTAACCTTCATCTCTTCCACCCATGTCTCTTTGAGCAATAAAGCCGATAACAAAATCTTTGTCGCTCTTCGCAATGTCAACAGTACCCTTAGTATATTCTCCAGTAGATAGGGAGCCCTTGCATGACAATTCTGCTAACATCAAAAGGCCTCTAGGTTCCTTTGTTACTTCTTCTGCCGCCTGCTTCAAACCGCTAACAATACCTGGGCCCACCACACCGTGTGCATTCGTAATGTCTGCCCATTCTGCTATTCTGTATACACCCGCAGAGTACTGCAATTTGACTGTATTACCAATGTCAGCAAATTTTCTGTCTTCGAAGAGTAAAAAATTGTACTTGGCGGATAATGCCTTTAGCGGCTTAACTGTGCCCTCCATGGAAAAATCAGTCAAGATATCCACATGTGTTTTTAGTAAACAAATTTTGGGACCTAATGCTTCAACTAACTCCAGTAATTCCTTGGTGGTACGAACATCCAATGAAGCACACAAGTTTGTTTGCTTTTCGTGCATGATATTAAATAGCTTGGCAGCAACAGGACTAGGATGAGTAGCAGCACGTTCCTTATATGTAGCTTTCGACATGATTTATCTTCGTTTCCTGCAGGTTTTTGTTCTGTGCAGTTGGGTTAAGAATACTGGGCAATTTCATGTTTCTTCAACACTACATATGCGTATATATACCAATCTAAGTCTGTGCTCCTTCCTTCGTTCTTCCTTCTGTTCGGAGATTACCGAATCAAAAAAATTTCAAGGAAACCGAAATCAAAAAAAAGAATAAAAAAAAAATGATGAATTGAAAAGCTTATCGATCCTACCCCTTGCGCTAAAGAAGTATATGTGCCTACTAACGCTTGTCTTTGTCTCTGTCACTAAACACTGGATTATTACTCCCAGATACTTATTTTGGACTAATTTAAATGATTTCGGATCAACGTTCTTAATATCGCTGAATCTTCCACAATTGATGAAAGTAGCTAGGAAGAGGAATTGGTATAAAGTTTTTGTTTTTGTAAATCTCGAAGTATACTCAAACGAATTTAGTATTTTCTCAGTGATCTCCCAGATGCTTTCACCCTCACTTAGAAGTGCTTTAAGCATTTTTTTACTGTGGCTATTTCCCTTATCTGCTTCTTCCGATGATTCGAACTGTAATTGCAAACTACTTACAATATCAGTGATATCAGATTGATGTTTTTGTCCATAGTAAGGAATAATTGTAAATTCCCAAGCAGGAATCAATTTCTTTAATGAGGCTTCCAGAATTGTTGCTTTTTGCGTCTTGTATTTAAACTGGAGTGATTTATTGACAATATCGAAACTCAGCGAATTGCTTATGATAGTATTATAGCTCATGAATGTGGCTCTCTTGATTGCTGTTCCGTTATGTGTAATCATCCAACATAAATAGGTTAGTTCAGCAGCACATAATGCTATTTTCTCACCTGAAGGTCTTTCAAACCTTTCCACAAACTGACGAACAAGCACCTTAGGTGGTGTTTTACATAATATATCAAATTGTGGCATGCTTAGCGCCGATCTTGTGTGCAATTGATATCTAGTTTCAACTACTCTATTTATCTTGTATCTTGCAGTATTCAAACACGCTAACTCGAAAAACTAACTTTAATTGTCCTGTTTGTCTCGCGTTCTTTCGAAAAATGCACCGGCCGCGCATTATTTGTACTGCGAAAATAATTGGTACTGCGGTATCTTCATTTCATATTTTAAAAATGCACCTTTGCTGCTTTTCCTTAATTTTTAGACGGCCCGCAGGTTCGTTTTGCGGTACTATCTTGTGATAAAAAGTTGTTTTGACATGTGATCTGCACAGATTTTATAATGTAATAAGCAAGAATACATTATCAAACGAACAATACTGGTAAAAGAAAACCAAAATGGACGACATTGAAACAGCCAAGAATCTGACGGTAAAAGCACGTACAGCTTATAGCGTCTGGGATGTATGTCGGCTGTTTATTGAAATGATTGCTCCTGATGTAGATATTGATATAGAGAGTAAACGTAAGTCTGATGAGCTACTCTTTCCAGGATATGTCATAAGGCCCATGGAATCTCTCACAACCGGTAGGCCGTATGGTCTTGATTCTAGCGCAGAAGATTCCAGCGTATCTTCTGACTCCAGTGCTGAGGTAATTTTGCCTGCTGCGAAGATGGTTAAGGAAAGGTTTGATTCGATTGGAAATGGTATGCTCTCTTCACAAGAAGCAAGTCAGGCTGCCATAGATTTGATGCTACAGAATAACAAGCTGTTAGACAATAGAAAGCAACTATACAAATCTATTGCTATAATAATAGGAAGATTGCCCGAGAAAGACAAGAAGAGAGCTACCGAAATGCTCATGAGAAAAATGGATTGTACACAGTTATTAGTCCCACCAGCTCCAACGGAAGAAGATGTTATGAAGCTCGTAAGCGTCGTTACCCAATTGCTTACTTTAGTTCCACCAGATCGTCAAGCTGCTTTAATAGGTGATTTATTCATCCCGGAATCTCTAAAGGATATATTCAATAGTTTCAATGAACTGGCGGCAGAGAATCGTTTACAGCAAAAAAAGAGTGAGTTGGAAGGAAGGACTGAAGTGAACCATGCTAATACAAATGAAGAAGTTCCCTCCAGGCGAACAAGAAGTAGAGACACAAATGCAAGAGGAGCATATAAATTACAAAACACCATCACTGAGGGCCCTAAAGCGGTTCCCACGAAAAAAAGGAGAGTAGCAACGAGGGTAAGGGGCAGAAAATCACGTAATACTTCTAGGGTATGATCCAATATCAAAGGAAATGATAGCATTGAAGGATGAGACTAATCCAATTGAGGAGTGGCAGCATATAGAACAGCTAAAGGGTAGTGCTGAAGGAAGCATACGATACCCCGCATGGAATGGGATAATATCACAGGAGGTACTAGACTACCTTTCATCCTACATAAATAGACGCATATAAGTACGCATTTAAGCATAAACACGCACTATGCCGTTCTTCTCATGTATATATATATACAGGCAACACGCAGATATAGGTGCGACGTGAACAGTGAGCTGTATGTGCGCAGCTCGCGTTGCATTTTCGGAAGCGCTCGTTTTCGGAAACGCTTTGAAGTTCCTATTCCGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCAGAGCGCTTTTGAAAACCAAAAGCGCTCTGAAGACGCACTTTCAAAAAACCAAAAACGCACCGGACTGTAACGAGCTACTAAAATATTGCGAATACCGCTTCCACAAACATTGCTCAAAAGTATCTCTTTGCTATATATCTCTGTGCTATATCCCTATATAACCTACCCATCCACCTTTCGCTCCTTGAACTTGCATCTAAACTCGACCTCTACATCAACAGGCTTCCAATGCTCTTCAAATTTTACTGTCAAGTAGACCCATACGGCTGTAATATGCTGCTCTTCATAATGTAAGCTTATCTTTATCGAATCGTGTGAAAAACTACTACCGCGATAAACCTTTACGGTTCCCTGAGATTGAATTAGTTCCTTTAGTATATGATACAAGACACTTTTGAACTTTGTACGACGAATTTTGAGGTTCGCCATCCTCTGGCTATTTCCAATTATCCTGTCGGCTATTATCTCCGCCTCAGTTTGATCTTCCGCTTCAGACTGCCATTTTTCACATAATGAATCTATTTCACCCCACAATCCTTCATCCGCCTCCGCATCTTGTTCCGTTAAACTATTGACTTCATGTTGTACATTGTTTAGTTCACGAGAAGGGTCCTCTTCAGGCGGTAGCTCCTGATCTCCTATATGACCTTTATCCTGTTCTCTTTCCACAAACTTAGAAATGTATTCATGAATTATGGAGCACCTAATAACATTCTTCAAGGCGGAGAAGTTTGGGCCAGATGCCCAATATGCTTGACATGAAAACGTGAGAATGAATTTAGTATTATTGTGATATTCTGAGGCAATTTTATTATAATCTCGAAGATAAGAGAAGAATGCAGTGACCTTTGTATTGACAAATGGAGATTCCATGTATCTAAAAAATACGCCTTTAGGCCTTCTGATACCCTTTCCCCTGCGGTTTAGCGTGCCTTTTACATTAATATCTAAACCCTCTCCGATGGTGGCCTTTAACTGACTAATAAATGCAACCGATATAAACTGTGATAATTCTGGGTGATTTATGATTCGATCGACAATTGTATTGTACACTAGTGCAGGATCAGGCCAATCCAGTTCTTTTTCAATTACCGGTGTGTCGTCTGTATTCAGTACATGTCCAACAAATGCAAATGCTAACGTTTTGTATTTCTTATAATTGTCAGGAACTGGAAAAGTCCCCCTTGTCGTCTCGATTACACACCTACTTTCATCGTACACCATAGGTTGGAAGTGCTGCATAATACATTGCTTAATACAAGCAAGCAGTCTCTCGCCATTCATATTTCAGTTATTTTCCATTACAGCTGATGTCATTGTATATCAGCGCTGTAAAAATCTATCTGTTACAGAAGGTTTTCGCGGTTTTTATAAACAAAACTTTCGTTACGAAATCGAGCAATCACCCCAGCTGCGTATTTGGAAATTCGGGAAAAAGTAGAGCAACGCGAGTTGCATTTTTTACACCATAATGCATGATTAACTTCGAGAAGGGATTAAGGCTAATTTCACTAGTATGTTTCAAAAACCTCAATCTGTCCATTGAATGCCTTATAAAACAGCTATAGATTGCATAGAAGAC.TTAGCTACTCAATGCTTTTTGTCAAAGCTTACTGATGATGATGTGTCTACTTTCAGGCGGGTCTGTAGTAAGGAGAATGACATTATAAAGCTGGCACTTAGAATTCCACGGACTATAGACTATACTAGTATACTCCGTCTACTGTACGATACACTTCCGCTCAGGTCCTTGTCCTTTAACGAGGCCTTACCACTCTTTTGTTACTCTATTGATCCAGCTCAGCAAAGGCAGTGTGATCTAAGATTCTATCTTCGCGATGTAGTAAAACTAGCTAGACCGAGAAAGAGACTAGAAATGCAAAAGGCACTTCTACAAT GGCTGCCATCATTATTATCCGATGTGACGCTGCAPolynucleotide sequence of Variant No. 73 yCDS: (SEQ ID NO: 8)TTGGATAACGGGTTAGCCCGTACACCTACTATGGGTTGGCTTCACTGGGAAAGATTCATGTGTAACTTAGATTGCCAAGAAGAGCCTGACAGCTGTATCTCAGAGAAACTATTCATGGAGATGGCTGAACTAATGGTAAGTGAAGGATGGAAGGATGCTGGTTATGAATACCTATGTATTGATGATTGCTGGATGGCTCCACAGCGTGATTCAGAAGGTAGGTTACAAGCTGACCCCCAGAGATTCCCACATGGCATACGTCAGCTTGCAAACTACGTACACAGCAAGGGTCTAAAGTTAGGCATCTACGCTGATGTCGGAAACAAGACATGTGCTGGTTTCCCAGGTTCATTCGGTTACTATGACATAGATGCGCAGACGTTTGCTGATTGGGGTGTTGATTTGTTGAAGTTTGATGGATGCTACTGCGATTCCCTGGAGAACCTAGCCGATGGGTACAAACACATGAGTTTGGCTCTAAACAGGACTGGTAGGAGCATCGTCTATAGTTGTGAATGGCCCTTGTACATGTGGCCGTTTCAGAAGCCAAACTACACTGAGATAAGACAATACTGTAACCATTGGCGTAACTTTGCTGACATAGATGATTCATGGGCTTCAATCAAATCTATCTTGGATTGGACTTCTTTCAACCAGGAAAGAATTGTTGATGTTGCAGGTCCAGGTGGATGGAATGACCCTGATATGCTTGTCATAGGGAACTTTGGGCTATCATGGAATCAACAAGTTACACAAATGGCTTTGTGGGCGATCATGGCCGCACCCCTATTCATGTCTAATGATCTACGTCACATATCACCCCAAGCAAAGGCTTTACTTCAAGATAAGGATGTCATAGCGATCAACCAAGATCCTCTTGGTAAACAAGGTTATCAATTGAGACAAGGTGACAACTTTGAAGTGTGGGAAAGACCATTGTCTGGACTTGCGTGGGCTGTTGCTATGATCAACCGTCAAGAGATCGGAGGGCCAAGATCTTACACTATCGCGGTAGCCTCTTTGGGTAAGGGTGTTGCGTGCAATCCTGCCTGCTTCATTACACAATTGCTTCCAGTTAAGAGAAAGTTGGGTTTCTATGAGTGGACATCTAGGCTAAGAAGTCACATCAATCCTACTGGTACGGTATTGTTGCAATTGGAGAACACAATGCAAATGTCTTTGAA AGATTTGTTAPolynucleotide sequence of Variant No. 73: (SEQ ID NO: 9)CTGGACAATGGATTGGCAAGGACGCCTACCATGGGCTGGCTGCACTGGGAGCGCTTCATGTGCAACCTTGACTGCCAGGAAGAGCCAGATTCCTGCATCAGTGAGAAGCTCTTCATGGAGATGGCAGAGCTCATGGTCTCAGAAGGCTGGAAGGATGCAGGTTATGAGTACCTCTGCATTGATGACTGTTGGATGGCTCCCCAAAGAGATTCAGAAGGCAGACTTCAGGCAGACCCTCAGCGCTTTCCTCATGGGATTCGCCAGCTAGCTAATTATGTTCACAGCAAAGGACTGAAGCTAGGGATTTATGCAGATGTTGGAAATAAAACCTGCGCAGGCTTCCCTGGGAGTTTTGGATACTACGACATTGATGCCCAGACCTTTGCTGACTGGGGAGTAGATCTGCTAAAATTTGATGGTTGTTACTGTGACAGTTTGGAAAATTTGGCAGATGGTTATAAGCACATGTCCTTGGCCCTGAATAGGACTGGCAGAAGCATTGTGTACTCCTGTGAGTGGCCTCTTTATATGTGGCCCTTTCAAAAGCCCAATTATACAGAAATCCGACAGTACTGCAATCACTGGCGAAATTTTGCTGACATTGATGATTCCTGGGCGAGTATAAAGAGTATCTTGGACTGGACATCTTTTAACCAGGAGAGAATTGTTGATGTTGCTGGACCAGGGGGTTGGAATGACCCAGATATGTTAGTGATTGGCAACTTTGGCCTCAGCTGGAATCAGCAAGTAACTCAGATGGCCCTCTGGGCTATCATGGCTGCTCCTTTATTCATGTCTAATGACCTCCGACACATCAGCCCTCAAGCCAAAGCTCTCCTTCAGGATAAGGACGTAATTGCCATCAATCAGGACCCCTTGGGCAAGCAAGGGTACCAGCTTAGACAGGGAGACAACTTTGAAGTGTGGGAACGACCTCTCTCAGGCTTAGCCTGGGCTGTAGCTATGATAAACCGGCAGGAGATTGGTGGACCTCGCTCTTATACCATCGCAGTTGCTTCCCTGGGTAAAGGAGTGGCCTGTAATCCTGCCTGCTTCATCACACAGCTCCTCCCTGTGAAAAGGAAGCTAGGGTTCTATGAATGGACTTCAAGGTTAAGAAGTCACATAAATCCCACAGGCACTGTTTTGCTTCAGCTAGAAAATACAATGCAGATGTCATTAAA AGACTTACTTPolypeptide sequence of Variant No. 73: (SEQ ID NO: 10)LDNGLARTPTMGWLHWERFMCNLDCQEEPDSCISEKLFMEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGNKTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKHMSLALNRTGRSIVYSCEWPLYMWPFQKPNYTEIRQYCNHWRNFADIDDSWASIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFGLSWNQQVTQMALWAIMAAPLFMSNDLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDNFEVWERPLSGLAWAVAMINRQEIGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTS RLRSHINPTGTVLLQLENTMQMSLKDLLPolynucleotide sequence of Variant No. 218 yCDS: (SEQ ID NO: 11)TTGGATAACGGGTTAGCCCGTACACCTACTATGGGTTGGCTTCACTGGGAAAGATTCATGTGTAACTTAGATTGCCAAGAAGAGCCTGACAGCTGTATCTCAGAGAAACTATTCATGGAGATGGCTGAACTAATGGTAAGTGAAGGATGGAAGGATGCTGGTTATGAATACCTATGTATTGATGATTGCTGGATGGCTCCACAGCGTGATTCAGAAGGTAGGTTACAAGCTGACCCCCAGAGATTCCCACATGGCATACGTCAGCTTGCAAACTACGTACACAGCAAGGGTCTAAAGTTAGGCATCTACGCTGATGTCGGAAACAAGACATGTGCTGGTTTCCCAGGTTCATTCGGTTACTATGACATAGATGCGCAGACGTTTGCTGATTGGGGTGTTGATTTGTTGAAGTTTGATGGATGCTACTGCGATTCCCTGGAGAACCTAGCCGATGGGTACAAACACATGAGTTTGGCTCTAAACAGGACTGGTAGGAGCATCGTCTATAGTTGTGAATGGCCCTTGTACATGTGGCCGTTTCAGAAGCCAAACTACACTGAGATAAGACAATACTGTAACCATTGGCGTAACTTTGCTGACATAGATGATTCATGGGCTTCAATCAAATCTATCTTGGATTGGACTTCTTTCAACCAGGAAAGAATTGTTGATGTTGCAGGTCCAGGTGGATGGAATGACCCTGATATGCTTGTCATAGGGAACTTTGGGCTATCATGGAATCAACAAGTTACACAAATGGCTTTGTGGGCGATCATGGCCGCACCCCTATTCATGTCTAATGATCTACGTCACATATCACCCCAAGCAAAGGCTTTACTTCAAGATAAGGATGTCATAGCGATCAACCAAGATCCTCTTGGTAAACAAGGTTATCAATTGAGACAAGGTGACAACTTTGAAGTGTGGGAAAGACCATTGTCTGGACTTGCGTGGGCTGTTGCTATTATCAACCGTCAAGAGATCGGAGGGCCAAGATCTTACACTATCGCGGTAGCCTCTTTGGGTAAGGGTGTTGCGTGCAATCCTGCCTGCTTCATTACACAATTGCTTCCAGTTAAGAGAAAGTTGGGTTTCTATAACTGGACATCTAGGCTAAAAAGTCACATTAATCCTACTGGTACGGTATTGTTGCAATTGGAGAACACAATGCAAATGTCTTTGAA AGATTTGTTAPolynucleotide sequence of Variant No. 218 hCDS: (SEQ ID NO: 12)CTGGACAATGGATTGGCAAGGACGCCTACCATGGGCTGGCTGCACTGGGAGCGCTTCATGTGCAACCTTGACTGCCAGGAAGAGCCAGATTCCTGCATCAGTGAGAAGCTCTTCATGGAGATGGCAGAGCTCATGGTCTCAGAAGGCTGGAAGGATGCAGGTTATGAGTACCTCTGCATTGATGACTGTTGGATGGCTCCCCAAAGAGATTCAGAAGGCAGACTTCAGGCAGACCCTCAGCGCTTTCCTCATGGGATTCGCCAGCTAGCTAATTATGTTCACAGCAAAGGACTGAAGCTAGGGATTTATGCAGATGTTGGAAATAAAACCTGCGCAGGCTTCCCTGGGAGTTTTGGATACTACGACATTGATGCCCAGACCTTTGCTGACTGGGGAGTAGATCTGCTAAAATTTGATGGTTGTTACTGTGACAGTTTGGAAAATTTGGCAGATGGTTATAAGCACATGTCCTTGGCCCTGAATAGGACTGGCAGAAGCATTGTGTACTCCTGTGAGTGGCCTCTTTATATGTGGCCCTTTCAAAAGCCCAATTATACAGAAATCCGACAGTACTGCAATCACTGGCGAAATTTTGCTGACATTGATGATTCCTGGGCGAGTATAAAGAGTATCTTGGACTGGACATCTTTTAACCAGGAGAGAATTGTTGATGTTGCTGGACCAGGGGGTTGGAATGACCCAGATATGTTAGTGATTGGCAACTTTGGCCTCAGCTGGAATCAGCAAGTAACTCAGATGGCCCTCTGGGCTATCATGGCTGCTCCTTTATTCATGTCTAATGACCTCCGACACATCAGCCCTCAAGCCAAAGCTCTCCTTCAGGATAAGGACGTAATTGCCATCAATCAGGACCCCTTGGGCAAGCAAGGGTACCAGCTTAGACAGGGAGACAACTTTGAAGTGTGGGAACGACCTCTCTCAGGCTTAGCCTGGGCTGTAGCTATTATAAACCGGCAGGAGATTGGTGGACCTCGCTCTTATACCATCGCAGTTGCTTCCCTGGGTAAAGGAGTGGCCTGTAATCCTGCCTGCTTCATCACACAGCTCCTCCCTGTGAAAAGGAAGCTAGGGTTCTATAACTGGACTTCAAGGTTAAAAAGTCACATAAATCCCACAGGCACTGTTTTGCTTCAGCTAGAAAATACAATGCAGATGTCATTAAA AGACTTACTTPolypeptide sequence of Variant No. 218: (SEQ ID NO: 13)LDNGLARTPTMGWLHWERFMCNLDCQEEPDSCISEKLFMEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGNKTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKHMSLALNRTGRSIVYSCEWPLYMWPFQKPNYTEIRQYCNHWRNFADIDDSWASIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFGLSWNQQVTQMALWAIMAAPLFMSNDLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDNFEVWERPLSGLAWAVAIINRQEIGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYNVVT SRLKSHINPTGTVLLQLENTMQMSLKDLLPolynucleotide sequence of Variant No. 326 yCDS: (SEQ ID NO: 14)TTGGATAACGGGTTAGCCCGTACACCTACTATGGGTTGGCTTCACTGGGAAAGATTCATGTGTAACTTAGATTGCCAAGAAGAGCCTGACAGCTGTATCTCAGAGAAACTATTCATGGAGATGGCTGAACGGATGGTAAGTGAAGGATGGAAGGATGCTGGTTATGAATACCTATGTATTGATGATTGCTGGATGGCTCCACAGCGTGATTCAGAAGGTAGGTTACAAGCTGACCCCCAGAGATTCCCACATGGCATACGTCAGCTTGCAAACTACGTACACAGCAAAGGTCTAAAGTTAGGCATCTACGCTGATGTCGGAAACAAGACATGTGCTGGTTTCCCAGGTTCATTCGGTTACTATGACATAGATGCGCAGACGTTTGCTGATTGGGGTGTTGATTTGTTGAAGTTTGATGGATGCTACTGCGATTCCCTGGAGAACCTAGCCGATGGGTACAAACACATGAGTTTGGCTCTAAACAGGACTGGTAGGAGCATCGTCTATAGTTGTGAATGGCCCTTGTACATGTGGCCGTTTCAGAAGCCAAACTACACTGAGATAAGACAATACTGTAACCATTGGCGTAACTTTGCTGACATAGATGATTCATGGGCTTCAATCAAATCTATCTTGGATTGGACTTCTCGTAACCAGGAAAGAATTGTTGATGTTGCAGGTCCAGGTGGATGGAATGACCCTGATATGCTTGTCATAGGGAACTTTGGGCTATCATGGGACCAACAAGTTACACAAATGGCTTTGTGGGCGATCATGGCCGCACCCCTATTCATGTCTAATGATCTACGTCACATATCACCCCAAGCAAAGGCTTTACTTCAAGATAAGGATGTCATAGCGATCAACCAAGATCCTCTTGGTAAACAAGGTTATCAATTGAGAAAAGGTGACAACTTTGAAGTGTGGGAAAGACCATTGTCTGGAGATGCGTGGGCTGTTGCTATTATCAACCGTCAAGAGATCGGAGGGCCAAGATCTTACACTATCCCGGTAGCCTCTTTGGGTAAGGGTGTTGCGTGCAATCCTGCCTGCTTCATTACACAATTGCTTCCAGTTAAGAGACAATTGGGTTTCTATAACTGGACCTCTAGGCTAAAAAGTCACATTAATCCTACTGGTACGGTATTGTTGCAATTGGAGAACACAATGCAAATGTCTTTGAA AGATTTGTTAPolypeptide sequence of Variant No. 326: (SEQ ID NO: 15)LDNGLARTPTMGWLHWERFMCNLDCQEEPDSCISEKLFMEMAERMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGNKTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKHMSLALNRTGRSIVYSCEWPLYMWPFQKPNYTEIRQYCNHWRNFADIDDSWASIKSILDWTSRNQERIVDVAGPGGWNDPDMLVIGNFGLSWDQQVTQMALWAIMAAPLFMSNDLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRKGDNFEVWERPLSGDAWAVAIINRQEIGGPRSYTIPVASLGKGVACNPACFITQLLPVKRQLGFYNVVTSRLKSHINPTGTVLLQLENTMQMSLK DLLPolynucleotide sequence of Variant No. 206 yCDS: (SEQ ID NO: 16)TTGGATAACGGGTTAGCCCGTACACCTACTATGGGTTGGCTTCACTGGGAAAGATTCATGTGTAACTTAGATTGCCAAGAAGAGCCTGACAGCTGTATCTCAGAGAAACTATTCATGGAGATGGCTGAACTAATGGTAAGTGAAGGATGGAAGGATGCTGGTTATGAATACCTATGTATTGATGATTGCTGGATGGCTCCACAGCGTGATTCAGAAGGTAGGTTACAAGCTGACCCCCAGAGATTCCCACATGGCATACGTCAGCTTGCAAACTACGTACACAGCAAGGGTCTAAAGTTAGGCATCTACGCTGATGTCGGAAACAAGACATGTGCTGGTTTCCCAGGTTCATTCGGTTACTATGACATAGATGCGCAGACGTTTGCTGATTGGGGTGTTGATTTGTTGAAGTTTGATGGATGCTACTGCGATTCCCTGGAGAACCTAGCCGATGGGTACAAACACATGAGTTTGGCTCTAAACAGGACTGGTAGGAGCATCGTCTATAGTTGTGAATGGCCCTTGTACATGTGGCCGTTTCAGAAGCCAAACTACACTGAGATAAGACAATACTGTAACCATTGGCGTAACTTTGCTGACATAGATGATTCATGGGCTTCAATCAAATCTATCTTGGATTGGACTTCTTTCAACCAGGAAAGAATTGTTGATGTTGCAGGTCCAGGTGGATGGAATGACCCTGATATGCTTGTCATAGGGAACTTTGGGCTATCATGGAATCAACAAGTTACACAAATGGCTTTGTGGGCGATCATGGCCGCACCCCTATTCATGTCTAATGATCTACGTCACATATCACCCCAAGCAAAGGCTTTACTTCAAGATAAGGATGTCATAGCGATCAACCAAGATCCTCTTGGTAAACAAGGTTATCAATTGAGACAAGGTGACAACTTTGAAGTGTGGGAAAGACCATTGTCTGGACTTGCGTGGGCTGTTGCTATGATCAACCGTCAAGAGATCGGAGGGCCAAGATCTTACACTATCGCGGTAGCCTCTTTGGGTAAGGGTGTTGCGTGCAATCCTGCCTGCTTCATTACACAATTGCTTCCAGTTAAGAGAAAGTTGGGTTTCTATAATTGGACCTCTAGGCTAAGAAGTCACATCAATCCTACTGGTACGGTATTGTTGCAATTGGAGAACACAATGCAAATGTCTTTGAAAGAT TTGTTAPolynucleotide sequence of Variant No. 206 hCDS: (SEQ ID NO: 17)CTGGACAATGGATTGGCAAGGACGCCTACCATGGGCTGGCTGCACTGGGAGCGCTTCATGTGCAACCTTGACTGCCAGGAAGAGCCAGATTCCTGCATCAGTGAGAAGCTCTTCATGGAGATGGCAGAGCTCATGGTCTCAGAAGGCTGGAAGGATGCAGGTTATGAGTACCTCTGCATTGATGACTGTTGGATGGCTCCCCAAAGAGATTCAGAAGGCAGACTTCAGGCAGACCCTCAGCGCTTTCCTCATGGGATTCGCCAGCTAGCTAATTATGTTCACAGCAAAGGACTGAAGCTAGGGATTTATGCAGATGTTGGAAATAAAACCTGCGCAGGCTTCCCTGGGAGTTTTGGATACTACGACATTGATGCCCAGACCTTTGCTGACTGGGGAGTAGATCTGCTAAAATTTGATGGTTGTTACTGTGACAGTTTGGAAAATTTGGCAGATGGTTATAAGCACATGTCCTTGGCCCTGAATAGGACTGGCAGAAGCATTGTGTACTCCTGTGAGTGGCCTCTTTATATGTGGCCCTTTCAAAAGCCCAATTATACAGAAATCCGACAGTACTGCAATCACTGGCGAAATTTTGCTGACATTGATGATTCCTGGGCGAGTATAAAGAGTATCTTGGACTGGACATCTTTTAACCAGGAGAGAATTGTTGATGTTGCTGGACCAGGGGGTTGGAATGACCCAGATATGTTAGTGATTGGCAACTTTGGCCTCAGCTGGAATCAGCAAGTAACTCAGATGGCCCTCTGGGCTATCATGGCTGCTCCTTTATTCATGTCTAATGACCTCCGACACATCAGCCCTCAAGCCAAAGCTCTCCTTCAGGATAAGGACGTAATTGCCATCAATCAGGACCCCTTGGGCAAGCAAGGGTACCAGCTTAGACAGGGAGACAACTTTGAAGTGTGGGAACGACCTCTCTCAGGCTTAGCCTGGGCTGTAGCTATGATAAACCGGCAGGAGATTGGTGGACCTCGCTCTTATACCATCGCAGTTGCTTCCCTGGGTAAAGGAGTGGCCTGTAATCCTGCCTGCTTCATCACACAGCTCCTCCCTGTGAAAAGGAAGCTAGGGTTCTATAACTGGACTTCAAGGTTAAGAAGTCACATAAATCCCACAGGCACTGTTTTGCTTCAGCTAGAAAATACAATGCAGATGTCATTAAAAGAC TTACTTPolypeptide sequence of Variant No. 206: (SEQ ID NO: 18)LDNGLARTPTMGWLHWERFMCNLDCQEEPDSCISEKLFMEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGNKTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKHMSLALNRTGRSIVYSCEWPLYMWPFQKPNYTEIRQYCNHWRNFADIDDSWASIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFGLSWNQQVTQMALWAIMAAPLFMSNDLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDNFEVWERPLSGLAWAVAMINRQEIGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYNWTSRLRSHINPTGTVLLQLENTMQMSLKD LLPolynucleotide sequence of Variant No. 205 yCDS: (SEQ ID NO: 19)TTGGATAACGGGTTAGCCCGTACACCTACTATGGGTTGGCTTCACTGGGAAAGATTCATGTGTAACTTAGATTGCCAAGAAGAGCCTGACAGCTGTATCTCAGAGAAACTATTCATGGAGATGGCTGAACTAATGGTAAGTGAAGGATGGAAGGATGCTGGTTATGAATACCTATGTATTGATGATTGCTGGATGGCTCCACAGCGTGATTCAGAAGGTAGGTTACAAGCTGACCCCCAGAGATTCCCACATGGCATACGTCAGCTTGCAAACTACGTACACAGCAAGGGTCTAAAGTTAGGCATCTACGCTGATGTCGGAAACAAGACATGTGCTGGTTTCCCAGGTTCATTCGGTTACTATGACATAGATGCGCAGACGTTTGCTGATTGGGGTGTTGATTTGTTGAAGTTTGATGGATGCTACTGCGATTCCCTGGAGAACCTAGCCGATGGGTACAAACACATGAGTTTGGCTCTAAACAGGACTGGTAGGAGCATCGTCTATAGTTGTGAATGGCCCTTGTACATGTGGCCGTTTCAGAAGCCAAACTACACTGAGATAAGACAATACTGTAACCATTGGCGTAACTTTGCTGACATAGATGATTCATGGGCTTCAATCAAATCTATCTTGGATTGGACTTCTTTCAACCAGGAAAGAATTGTTGATGTTGCAGGTCCAGGTGGATGGAATGACCCTGATATGCTTGTCATAGGGAACTTTGGGCTATCATGGAATCAACAAGTTACACAAATGGCTTTGTGGGCGATCATGGCCGCACCCCTATTCATGTCTAATGATCTACGTCACATATCACCCCAAGCAAAGGCTTTACTTCAAGATAAGGATGTCATAGCGATCAACCAAGATCCTCTTGGTAAACAAGGTTATCAATTGAGACAAGGTGACAACTTTGAAGTGTGGGAAAGACCATTGTCTGGACTTGCGTGGGCTGTTGCTATGATCAACCGTCAAGAGATCGGAGGGCCAAGATCTTACACTATCGCGGTAGCCTCTTTGGGTAAGGGTGTTGCGTGCAATCCTGCCTGCTTCATTACACAATTGCTTCCAGTTAAGAGAAAGTTGGGTTTCTATGATTGGGACTCTAGGCTAAGAAGTCACATCAATCCTACTGGTACGGTATTGTTGCAATTGGAGAACACAATGCAAATGTCTTTGAAAGAT TTGTTAPolynucleotide sequence of Variant No. 205 hCDS: (SEQ ID NO: 20)CTGGACAATGGATTGGCAAGGACGCCTACCATGGGCTGGCTGCACTGGGAGCGCTTCATGTGCAACCTTGACTGCCAGGAAGAGCCAGATTCCTGCATCAGTGAGAAGCTCTTCATGGAGATGGCAGAGCTCATGGTCTCAGAAGGCTGGAAGGATGCAGGTTATGAGTACCTCTGCATTGATGACTGTTGGATGGCTCCCCAAAGAGATTCAGAAGGCAGACTTCAGGCAGACCCTCAGCGCTTTCCTCATGGGATTCGCCAGCTAGCTAATTATGTTCACAGCAAAGGACTGAAGCTAGGGATTTATGCAGATGTTGGAAATAAAACCTGCGCAGGCTTCCCTGGGAGTTTTGGATACTACGACATTGATGCCCAGACCTTTGCTGACTGGGGAGTAGATCTGCTAAAATTTGATGGTTGTTACTGTGACAGTTTGGAAAATTTGGCAGATGGTTATAAGCACATGTCCTTGGCCCTGAATAGGACTGGCAGAAGCATTGTGTACTCCTGTGAGTGGCCTCTTTATATGTGGCCCTTTCAAAAGCCCAATTATACAGAAATCCGACAGTACTGCAATCACTGGCGAAATTTTGCTGACATTGATGATTCCTGGGCGAGTATAAAGAGTATCTTGGACTGGACATCTTTTAACCAGGAGAGAATTGTTGATGTTGCTGGACCAGGGGGTTGGAATGACCCAGATATGTTAGTGATTGGCAACTTTGGCCTCAGCTGGAATCAGCAAGTAACTCAGATGGCCCTCTGGGCTATCATGGCTGCTCCTTTATTCATGTCTAATGACCTCCGACACATCAGCCCTCAAGCCAAAGCTCTCCTTCAGGATAAGGACGTAATTGCCATCAATCAGGACCCCTTGGGCAAGCAAGGGTACCAGCTTAGACAGGGAGACAACTTTGAAGTGTGGGAACGACCTCTCTCAGGCTTAGCCTGGGCTGTAGCTATGATAAACCGGCAGGAGATTGGTGGACCTCGCTCTTATACCATCGCAGTTGCTTCCCTGGGTAAAGGAGTGGCCTGTAATCCTGCCTGCTTCATCACACAGCTCCTCCCTGTGAAAAGGAAGCTAGGGTTCTATGATTGGGATTCAAGGTTAAGAAGTCACATAAATCCCACAGGCACTGTTTTGCTTCAGCTAGAAAATACAATGCAGATGTCATTAAAAGAC TTACTTPolypeptide sequence of Variant No. 205: (SEQ ID NO: 21)LDNGLARTPTMGWLHWERFMCNLDCQEEPDSCISEKLFMEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGNKTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKHMSLALNRTGRSIVYSCEWPLYMWPFQKPNYTEIRQYCNHWRNFADIDDSWASIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFGLSWNQQVTQMALWAIMAAPLFMSNDLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDNFEVWERPLSGLAWAVAMINRQEIGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYDWDSRLRSHINPTGTVLLQLENTMQMSLKD LLPolynucleotide sequence of Variant No. 76 yCDS: (SEQ ID NO: 22)TTGGATAACGGGTTAGCCCGTACACCTACTATGGGTTGGCTTCACTGGGAAAGATTCATGTGTAACTTAGATTGCCAAGAAGAGCCTGACAGCTGTATCTCAGAGAAACTATTCATGGAGATGGCTGAACTAATGGTAAGTGAAGGATGGAAGGATGCTGGTTATGAATACCTATGTATTGATGATTGCTGGATGGCTCCACAGCGTGATTCAGAAGGTAGGTTACAAGCTGACCCCCAGAGATTCCCACATGGCATACGTCAGCTTGCAAACTACGTACACAGCAAGGGTCTAAAGTTAGGCATCTACGCTGATGTCGGAAACAAGACATGTGCTGGTTTCCCAGGTTCATTCGGTTACTATGACATAGATGCGCAGACGTTTGCTGATTGGGGTGTTGATTTGTTGAAGTTTGATGGATGCTACTGCGATTCCCTGGAGAACCTAGCCGATGGGTACAAACACATGAGTTTGGCTCTAAACAGGACTGGTAGGAGCATCGTCTATAGTTGTGAATGGCCCTTGTACATGTGGCCGTTTCAGAAGCCAAACTACACTGAGATAAGACAATACTGTAACCATTGGCGTAACTTTGCTGACATAGATGATTCATGGAGGTCAATCAAATCTATCTTGGATTGGACTTCTTTCAACCAGGAAAGAATTGTTGATGTTGCAGGTCCAGGTGGATGGAATGACCCTGATATGCTTGTCATAGGGAACTTTGGGCTATCATGGAATCAACAAGTTACACAAATGGCTTTGTGGGCGATCATGGCCGCACCCCTATTCATGTCTAATGATCTACGTCACATATCACCCCAAGCAAAGGCTTTACTTCAAGATAAGGATGTCATAGCGATCAACCAAGATCCTCTTGGTAAACAAGGTTATCAATTGAGACAAGGTGACAACTTTGAAGTGTGGGAAAGACCATTGTCTGGACTTGCGTGGGCTGTTGCTATGATCAACCGTCAAGAGATCGGAGGGCCAAGATCTTACACTATCGCGGTAGCCTCTTTGGGTAAGGGTGTTGCGTGCAATCCTGCCTGCTTCATTACACAATTGCTTCCAGTTAAGAGAAAGTTGGGTTTCTATGAGTGGACATCTAGGCTAAGAAGTCACATCAATCCTACTGGTACGGTATTGTTGCAATTGGAGAACACAATGCAAATGTCTTTGAAAGAT TTGTTAPolynucleotide sequence of Variant No. 76 hCDS: (SEQ ID NO: 23)CTGGACAATGGATTGGCAAGGACGCCTACCATGGGCTGGCTGCACTGGGAGCGCTTCATGTGCAACCTTGACTGCCAGGAAGAGCCAGATTCCTGCATCAGTGAGAAGCTCTTCATGGAGATGGCAGAGCTCATGGTCTCAGAAGGCTGGAAGGATGCAGGTTATGAGTACCTCTGCATTGATGACTGTTGGATGGCTCCCCAAAGAGATTCAGAAGGCAGACTTCAGGCAGACCCTCAGCGCTTTCCTCATGGGATTCGCCAGCTAGCTAATTATGTTCACAGCAAAGGACTGAAGCTAGGGATTTATGCAGATGTTGGAAATAAAACCTGCGCAGGCTTCCCTGGGAGTTTTGGATACTACGACATTGATGCCCAGACCTTTGCTGACTGGGGAGTAGATCTGCTAAAATTTGATGGTTGTTACTGTGACAGTTTGGAAAATTTGGCAGATGGTTATAAGCACATGTCCTTGGCCCTGAATAGGACTGGCAGAAGCATTGTGTACTCCTGTGAGTGGCCTCTTTATATGTGGCCCTTTCAAAAGCCCAATTATACAGAAATCCGACAGTACTGCAATCACTGGCGAAATTTTGCTGACATTGATGATTCCTGGCGTAGTATAAAGAGTATCTTGGACTGGACATCTTTTAACCAGGAGAGAATTGTTGATGTTGCTGGACCAGGGGGTTGGAATGACCCAGATATGTTAGTGATTGGCAACTTTGGCCTCAGCTGGAATCAGCAAGTAACTCAGATGGCCCTCTGGGCTATCATGGCTGCTCCTTTATTCATGTCTAATGACCTCCGACACATCAGCCCTCAAGCCAAAGCTCTCCTTCAGGATAAGGACGTAATTGCCATCAATCAGGACCCCTTGGGCAAGCAAGGGTACCAGCTTAGACAGGGAGACAACTTTGAAGTGTGGGAACGACCTCTCTCAGGCTTAGCCTGGGCTGTAGCTATGATAAACCGGCAGGAGATTGGTGGACCTCGCTCTTATACCATCGCAGTTGCTTCCCTGGGTAAAGGAGTGGCCTGTAATCCTGCCTGCTTCATCACACAGCTCCTCCCTGTGAAAAGGAAGCTAGGGTTCTATGAATGGACTTCAAGGTTAAGAAGTCACATAAATCCCACAGGCACTGTTTTGCTTCAGCTAGAAAATACAATGCAGATGTCATTAAAAGAC TTACTTPolypeptide sequence of Variant No. 76: (SEQ ID NO: 24)LDNGLARTPTMGWLHWERFMCNLDCQEEPDSCISEKLFMEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGNKTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKHMSLALNRTGRSIVYSCEWPLYMWPFQKPNYTEIRQYCNHWRNFADIDDSWRSIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFGLSWNQQVTQMALWAIMAAPLFMSNDLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDNFEVWERPLSGLAWAVAMINRQEIGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSHINPTGTVLLQLENTMQMSLKD LLPolynucleotide sequence of Mfalpha signal peptide: (SEQ ID NO: 25)ATGAGATTTCCTTCAATTTTTACTGCAGTTTTATTC GCAGCATCCTCCGCATTAGCTPolypeptide sequence of Mfalpha signal peptide: (SEQ ID NO: 26)MRFPSIFTAVLFAASSALA Polynucleotide sequence of MMO435: (SEQ ID NO: 27)ttaactatatcgtaatacacaggatccaccATGA GATTTCCTTCAATTTTTACTGPolynucleotide sequence of MMO439: (SEQ ID NO :28)AGTAGGTGTACGGGCTAACCCGTTATCCAAAGCT AATGCGGAGGATGCPolynucleotide sequence of MMO514: (SEQ ID NO: 29)TTTTACTGCAGTTTTATTCGCAGCATCCTCCGCA TTAGCTTTGGATAACGGGTTAGCCCGPolynucleotide sequence of MMO481: (SEQ ID NO: 30)GAGCTAAAAGTACAGTGGGAACAAAGTCGAGGTC GACTTATAACAAATCTTTCAAAGACAPolynucleotide sequence of Synthetic mammalian signal peptide:(SEQ ID NO: 31) ATGGAATGGAGCTGGGTCTTTCTCTTCTTCCTGTCAGTAACGACTGGTGTCCACTCC Polynucleotide sequence of LAKE Fw:(SEQ ID NO: 32) CGATCGAAGCTTCGCCACCAPolynucleotide sequence of Br reverse: (SEQ ID NO: 33)CTTGCCAATCCATTGTCCAGGGAGTGGACACCAGT CGTTAPolynucleotide sequence of Br Fw: (SEQ ID NO: 34)TAACGACTGGTGTCCACTCCCTGGACAATGGATT GGCAAGPolynucleotide sequence of hGLA Rv: (SEQ ID NO: 35)CGATCGGCGGCCGCTCAAAGTAAGTCTTTTAATGACA Polynucleotide sequence ofSP-GLA (yCDS): (SEQ ID NO: 36) ATGAGATTTCCTTCAATTTTTACTGCAGTTTTATTCGCAGCATCCTCCGCATTAGCTTTGGATAACGGGTTAGCCCGTACACCTACTATGGGTTGGCTTCACTGGGAAAGATTCATGTGTAACTTAGATTGCCAAGAAGAGCCTGACAGCTGTATCTCAGAGAAACTATTCATGGAGATGGCTGAACTAATGGTAAGTGAAGGATGGAAGGATGCTGGTTATGAATACCTATGTATTGATGATTGCTGGATGGCTCCACAGCGTGATTCAGAAGGTAGGTTACAAGCTGACCCCCAGAGATTCCCACATGGCATACGTCAGCTTGCAAACTACGTACACAGCAAGGGTCTAAAGTTAGGCATCTACGCTGATGTCGGAAACAAGACATGTGCTGGTTTCCCAGGTTCATTCGGTTACTATGACATAGATGCGCAGACGTTTGCTGATTGGGGTGTTGATTTGTTGAAGTTTGATGGATGCTACTGCGATTCCCTGGAGAACCTAGCCGATGGGTACAAACACATGAGTTTGGCTCTAAACAGGACTGGTAGGAGCATCGTCTATAGTTGTGAATGGCCCTTGTACATGTGGCCGTTTCAGAAGCCAAACTACACTGAGATAAGACAATACTGTAACCATTGGCGTAACTTTGCTGACATAGATGATTCATGGAAGTCAATCAAATCTATCTTGGATTGGACTTCTTTCAACCAGGAAAGAATTGTTGATGTTGCAGGTCCAGGTGGATGGAATGACCCTGATATGCTTGTCATAGGGAACTTTGGGCTATCATGGAATCAACAAGTTACACAAATGGCTTTGTGGGCGATCATGGCCGCACCCCTATTCATGTCTAATGATCTACGTCACATATCACCCCAAGCAAAGGCTTTACTTCAAGATAAGGATGTCATAGCGATCAACCAAGATCCTCTTGGTAAACAAGGTTATCAATTGAGACAAGGTGACAACTTTGAAGTGTGGGAAAGACCATTGTCTGGACTTGCGTGGGCTGTTGCTATGATCAACCGTCAAGAGATCGGAGGGCCAAGATCTTACACTATCGCGGTAGCCTCTTTGGGTAAGGGTGTTGCGTGCAATCCTGCCTGCTTCATTACACAATTGCTTCCAGTTAAGAGAAAGTTGGGTTTCTATGAGTGGACATCTAGGCTAAGAAGTCACATCAATCCTACTGGTACGGTATTGTTGCAATTGGAGAAC ACAATGCAAATGTCTTTGAAAGATTTGTTAPolynucleotide Sequence of MFleader- GLA (yCDS): (SEQ ID NO: 37)ATGAGATTTCCTTCAATTTTTACTGCAGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTCCAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTACTTAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAACGGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTATCTTTGGATAAAAGATTGGATAACGGGTTAGCCCGTACACCTACTATGGGTTGGCTTCACTGGGAAAGATTCATGTGTAACTTAGATTGCCAAGAAGAGCCTGACAGCTGTATCTCAGAGAAACTATTCATGGAGATGGCTGAACTAATGGTAAGTGAAGGATGGAAGGATGCTGGTTATGAATACCTATGTATTGATGATTGCTGGATGGCTCCACAGCGTGATTCAGAAGGTAGGTTACAAGCTGACCCCCAGAGATTCCCACATGGCATACGTCAGCTTGCAAACTACGTACACAGCAAGGGTCTAAAGTTAGGCATCTACGCTGATGTCGGAAACAAGACATGTGCTGGTTTCCCAGGTTCATTCGGTTACTATGACATAGATGCGCAGACGTTTGCTGATTGGGGTGTTGATTTGTTGAAGTTTGATGGATGCTACTGCGATTCCCTGGAGAACCTAGCCGATGGGTACAAACACATGAGTTTGGCTCTAAACAGGACTGGTAGGAGCATCGTCTATAGTTGTGAATGGCCCTTGTACATGTGGCCGTTTCAGAAGCCAAACTACACTGAGATAAGACAATACTGTAACCATTGGCGTAACTTTGCTGACATAGATGATTCATGGAAGTCAATCAAATCTATCTTGGATTGGACTTCTTTCAACCAGGAAAGAATTGTTGATGTTGCAGGTCCAGGTGGATGGAATGACCCTGATATGCTTGTCATAGGGAACTTTGGGCTATCATGGAATCAACAAGTTACACAAATGGCTTTGTGGGCGATCATGGCCGCACCCCTATTCATGTCTAATGATCTACGTCACATATCACCCCAAGCAAAGGCTTTACTTCAAGATAAGGATGTCATAGCGATCAACCAAGATCCTCTTGGTAAACAAGGTTATCAATTGAGACAAGGTGACAACTTTGAAGTGTGGGAAAGACCATTGTCTGGACTTGCGTGGGCTGTTGCTATGATCAACCGTCAAGAGATCGGAGGGCCAAGATCTTACACTATCGCGGTAGCCTCTTTGGGTAAGGGTGTTGCGTGCAATCCTGCCTGCTTCATTACACAATTGCTTCCAGTTAAGAGAAAGTTGGGTTTCTATGAGTGGACATCTAGGCTAAGAAGTCACATCAATCCTACTGGTACGGTATTGTTGCAATTGGAGAACACAATGCAAATGTCTTTGAAAGAT TTGTTAPolypeptide Sequence of MFleader: (SEQ ID NO: 38)MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIA AKEEGVSLDKRPolynucleotide sequence of Variant No. 395 yCDS: (SEQ ID NO: 39)TTGGATAACGGGTTAGCCCGTACACCTACTATGGGTTGGCTTCACTGGGAAAGATTCATGTGTAACTTAGATTGCCAAGAAGAGCCTGACAGCTGTATCTCAGAGAAACTATTCATGGAGATGGCTGAACGGATGGTAAGTGAAGGATGGAAGGATGCTGGTTATGAATACCTATGTATTGATGATTGCTGGATGGCTCCACAGCGTGATTCAGAAGGTAGGTTACAAGCTGACCCCCAGAGATTCCCACATGGCATACGTCAGCTTGCAAACCATGTACACAGCAAAGGTCTAAAGTTAGGCATCTACGCTGATGTCGGAAACAAGACATGTGCTGGTTTCCCAGGTTCATTCGGTTACTATGACATAGATGCGCAGACGTTTGCTGATTGGGGTGTTGATTTGTTGAAGTTTGATGGATGCTACTGCGATTCCCTGGAGAACCTAGCCGATGGGTACAAACACATGAGTTTGGCTCTAAACAGGACTGGTAGGAGCATCGTCTATAGTTGTGAATGGCCCTTGTACATGTGGCCGTTTCAGAAGCCAAACTACACTGAGATAAGACAATACTGTAACCATTGGCGTAACTTTGCTGACATAGATGATTCATGGGCTTCAATCAAATCTATCTTGGATTGGACTTCTCGTAACCAGGAAAGAATTGTTGATGTTGCAGGTCCAGGTGGATGGAATGACCCTGATATGCTTGTCATAGGGAACTTTGGGCTATCATGGGACCAACAAGTTACACAAATGGCTTTGTGGGCGATCATGGCCGCACCCCTATTCATGTCTAATGATCTACGTCACATATCACCCCAAGCAAAGGCTTTACTTCAAGATAAGGATGTCATAGCGATCAACCAAGATCCTCTTGGTAAACAAGGTTATCAATTGAGAAAAGGTGACAACTTTGAAGTGTGGGAAAGACCATTGTCTGGAGATGCGTGGGCTGTTGCTATTATCAACCGTCAAGAGATCGGAGGGCCAAGATCTTACACTATCCCGGTAGCCTCTTTGGGTAAGGGTGTTGCGTGCAATCCTGCCTGCTTCATTACACAATTGCTTCCAGTTAAGAGACAATTGGGTTTCTATAACTGGACCTCTAGGCTAAAAAGTCACATTAATCCTACTGGTACGGTATTGTTGCAATTGGAGAACACAATGCAAATGTCTTTGAA AGATTTGTTAPolypeptide sequence of Variant No. 395: (SEQ ID NO: 40)LDNGLARTPTMGWLHWERFMCNLDCQEEPDSCISEKLFMEMAERMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANHVHSKGLKLGIYADVGNKTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKHMSLALNRTGRSIVYSCEWPLYMWPFQKPNYTEIRQYCNHWRNFADIDDSWASIKSILDWTSRNQERIVDVAGPGGWNDPDMLVIGNFGLSWDQQVTQMALWAIMAAPLFMSNDLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRKGDNFEVWERPLSGDAWAVAIINRQEIGGPRSYTIPVASLGKGVACNPACFITQLLPVKRQLGFYNVVT SRLKSHINPTGTVLLQLENTMQMSLKDLLPolynucleotide sequence of Variant No. 402 yCDS: (SEQ ID NO: 41)TTGGATAACGGGTTAGCCCGTACACCTACTATGGGTTGGCTTCACTGGGAAAGATTCATGTGTAACTTAGATTGCCAAGAAGAGCCTGACAGCTGTATCTCAGAGAAACTATTCATGGAGATGGCTGAACGGATGGTAAGTGAAGGATGGAAGGATGCTGGTTATGAATACCTATGTATTGATGATTGCTGGATGGCTCCACAGCGTGATTCAGAAGGTAGGTTACAAGCTGACCCCCAGAGATTCCCACATGGCATACGTCAGCTTGCAAACTACGTACACAGCAAAGGTCTAAAGTTAGGCATCTACGCTGATGTCGGAAACAAGACATGTGCTGGTTTCCCAGGTTCATTCGGTTACTATGACATAGATGCGCAGACGTTTGCTGATTGGGGTGTTGATTTGTTGAAGTTTGATGGATGCTACTGCGATTCCCTGGAGAACCTAGCCGATGGGTACAAACACATGAGTTTGGCTCTAAACAGGACTGGTAGGCCGATCGTCTATAGTTGTGAATGGCCCTTGTACATGTGGCCGTTTCAGAAGCCAAACTACACTGAGATAAGACAATACTGTAACCATTGGCGTAACTTTGCTGACATAGATGATTCATGGGCTTCAATCAAATCTATCTTGGATTGGACTTCTCGTAACCAGGAAAGAATTGTTGATGTTGCAGGTCCAGGTGGATGGAATGACCCTGATATGCTTGTCATAGGGAACTTTGGGCTATCATGGGACCAACAAGTTACACAAATGGCTTTGTGGGCGATCATGGCCGCACCCCTATTCATGTCTAATGATCTACGTCACATATCACCCCAAGCAAAGGCTTTACTTCAAGATAAGGATGTCATAGCGATCAACCAAGATCCTCTTGGTAAACAAGGTTATCAATTGAGAAAAGGTGACAACTTTGAAGTGTGGGAAAGACCATTGTCTGGAGATGCGTGGGCTGTTGCTATTATCAACCGTCAAGAGATCGGAGGGCCAAGATCTTACACTATCCCGGTAGCCTCTTTGGGTAAGGGTGTTGCGTGCAATCCTGCCTGCTTCATTACACAATTGCTTCCAGTTAAGAGACAATTGGGTTTCTATAACTGGACCTCTAGGCTAAAAAGTCACATTAATCCTACTGGTACGGTATTGTTGCAATTGGAGAACACAATGCAAATGTCTTTGAA AGATTTGTTAPolypeptide sequence of Variant No. 402: (SEQ ID NO: 42)LDNGLARTPTMGWLHWERFMCNLDCQEEPDSCISEKLFMEMAERMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGNKTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKHMSLALNRTGRPIVYSCEWPLYMWPFQKPNYTEIRQYCNHWRNFADIDDSWASIKSILDWTSRNQERIVDVAGPGGWNDPDMLVIGNFGLSWDQQVTQMALWAIMAAPLFMSNDLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRKGDNFEVWERPLSGDAWAVAIINRQEIGGPRSYTIPVASLGKGVACNPACFITQLLPVKRQLGFYNVVT SRLKSHINPTGTVLLQLENTMQMSLKDLLPolynucleotide sequence of Variant No. 625 yCDS: (SEQ ID NO: 43)TTGGATAACGGGTTAGCCCGTACACCTACTATGGGTTGGCTTCACTGGGAAAGATTCATGTGTAACTTAGATTGCCAAGAAGAGCCTGACAGCTGTATCTCAGAGAAACTATTCATGGAGATGGCTGAACGGATGGTAACCGAAGGATGGAAGGATGCTGGTTATGAATACCTATGTATTGATGATTGCTGGATGGCTCCACAGCGTGATTCAGAAGGTAGGTTACAAGCTGACCCCCAGAGATTCCCACATGGCATACGTCAGCTTGCAAACCATGTACACAGCAAAGGTCTAAAGTTAGGCATCTACGCTGATGTCGGAAACAAGACATGTGCTGGTTTCCCAGGTTCATTCGGTTACTATGACATAGATGCGCAGACGTTTGCTGATTGGGGTGTTGATTTGTTGAAGTTTGATGGATGCTACTGCGATTCCCTGGAGAACCTAGCCGATGGGTACAAACACATGAGTTTGGCTCTAAACAGGACTGGTAGGCCGATCGTCTATAGTTGTGAATGGCCCTTGTACATGTGGCCGTTTCAGAAGCCAAACTACACTGAGATAAGACAATACTGTAACCATTGGCGTAACTTTGCTGACATAGATGATTCATGGGCTTCAATCAAATCTATCTTGGATTGGACTTCTCGTAACCAGGAAAGAATTGTTGATGTTGCAGGTCCAGGTGGATGGAATGACCCTGATATGCTTGTCATAGGGAACTTTGGGCTATCATGGGACCAACAAGTTACACAAATGGCTTTGTGGGCGATCATGGCCGCACCCCTATTCATGTCTAATGATCTACGTGCGATATCACCCCAAGCAAAGGCTTTACTTCAAGATAAGGATGTCATAGCGATCAACCAAGATCCTCTTGGTAAACAAGGTTATCAATTGAGAAAAGGTGACAACTTTGAAGTGTGGGAAAGACCATTGTCTGGAGATGCGTGGGCTGTTGCTATTATCAACCGTCAAGAGATCGGAGGGCCAAGATCTTACACTATCCCGGTAGCCTCTTTGGGTAAGGGTGTTGCGTGCAATCCTGCCTGCTTCATTACACAATTGCTTCCAGTTAAGAGACAATTGGGTTTCTATAACTGGACCTCTAGGCTAAAAAGTCACATTAATCCTACTGGTACGGTATTGTTGCAATTGGAGAACACAATGCAAACCTCTTTGAA AGATTTGTTAPolypeptide sequence of Variant No. 625: (SEQ ID NO: 44)LDNGLARTPTMGWLHWERFMCNLDCQEEPDSCISEKLFMEMAERMVTEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANHVHSKGLKLGIYADVGNKTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKHMSLALNRTGRPIVYSCEWPLYMWPFQKPNYTEIRQYCNHWRNFADIDDSWASIKSILDWTSRNQERIVDVAGPGGWNDPDMLVIGNFGLSWDQQVTQMALWAIMAAPLFMSNDLRAISPQAKALLQDKDVIAINQDPLGKQGYQLRKGDNFEVWERPLSGDAWAVAIINRQEIGGPRSYTIPVASLGKGVACNPACFITQLLPVKRQLGFYNVVT SRLKSHINPTGTVLLQLENTMQTSLKDLLPolynucleotide sequence of Variant No. 648 yCDS: (SEQ ID NO: 45)TTGGATAACGGGTTAGCCCGTACACCTCCGATGGGTTGGCTTCACTGGGAAAGATTCATGTGTAACTTAGATTGCCAAGAAGAGCCTGACAGCTGTATCTCAGAGAAACTATTCGAAGAGATGGCTGAACGGATGGTAACCGAAGGATGGAAGGATGCTGGTTATGAATACCTATGTATTGATGATTGCTGGATGGCTCCACAGCGTGATTCAGAAGGTAGGTTACAAGCTGACCCCCAGAGATTCCCACATGGCATACGTCAGCTTGCAAACCATGTACACAGCAAAGGTCTAAAGTTAGGCATCTACGCTGATGTCGGAAACAAGACATGTGCTGGTTTCCCAGGTTCATTCGGTTACTATGACATAGATGCGCAGACGTTTGCTGATTGGGGTGTTGATTTGTTGAAGTTTGATGGATGCTACTGCGATTCCCTGGAGAACCTAGCCGATGGGTACAAACACATGAGTTTGGCTCTAAACAGGACTGGTAGGCCGATCGTCTATAGTTGTGAATGGCCCTTGTACATGTGGCCGTTTCAGAAGCCAAACTACACTGAGATAAGACAATACTGTAACCATTGGCGTAACTTTGCTGACATAGATGATTCATGGGCTTCAATCAAATCTATCTTGGATTGGACTTCTCGTAACCAGGAAAGAATTGTTGATGTTGCAGGTCCAGGTGGATGGAATGACCCTGATATGCTTGTCATAGGGAACTTTGGGCTATCATGGGACCAACAAGTTACACAAATGGCTTTGTGGGCGATCATGGCCGGCCCCCTATTCATGTCTAATGATCTACGTGCGATATCACCCCAAGCAAAGGCTTTACTTCAAGATAAGGATGTCATAGCGATCAACCAAGATCCTCTTGGTAAACAAGGTTATCAATTGAGAAAAGGTGACAACTTTGAAGTGTGGGAAAGACCATTGTCTGGAGATGCGTGGGCTGTTGCTATTATCAACCGTCAAGAGATCGGAGGGCCAAGATCTTACACTATCCCGGTAGCCTCTTTGGGTAAGGGTGTTGCGTGCAATCCTGCCTGCTTCATTACACAATTGCTTCCAGTTAAGAGACAATTGGGTTTCTATAACGCAACCTCTAGGCTAAAAAGTCACATTAATCCTACTGGTACGGTATTGTTGCAATTGGAGAACACAATGCAAACCTCTTTGAA AGATTTGTTAPolypeptide sequence of Variant No. 648: (SEQ ID NO: 46)LDNGLARTPPMGWLHWERFMCNLDCQEEPDSCISEKLFEEMAERMVTEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANHVHSKGLKLGIYADVGNKTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKHMSLALNRTGRPIVYSCEWPLYMWPFQKPNYTEIRQYCNHWRNFADIDDSWASIKSILDWTSRNQERIVDVAGPGGWNDPDMLVIGNFGLSWDQQVTQMALWAIMAGPLFMSNDLRAISPQAKALLQDKDVIAINQDPLGKQGYQLRKGDNFEVWERPLSGDAWAVAIINRQEIGGPRSYTIPVASLGKGVACNPACFITQLLPVKRQLGFYNATS RLKSHINPTGTVLLQLENTMQTSLKDLL

Example 1 GLA Gene Acquisition and Construction of Expression Vectors

A synthetic gene coding for a WT human GLA was designed for optimizedgene expression in Saccharomyces cerevisiae (SEQ ID NO:3), assembled,and subcloned into the E. coli expression vector pCK100900i (SEQ IDNO:6).

A chimeric GLA expression construct encoding a 19 amino acid S.cerevisae MFalpha signal peptide fused to the mature form ofyeast-optimized GLA was generated in a yeast expression vector designedfor secreted expression, as follows. A fragment coding for the MFalphasignal peptide (SEQ ID NO:25) was amplified by PCR using theoligonucleotides MMO435 (SEQ ID NO:27)and MMO439 (SEQ ID NO:28) fromS288C genomic DNA, and a fragment coding for a synthetic GLA (SEQ IDNO:3) was amplified using primers MMO514 (SEQ ID NO:29) and MMO481 (SEQID NO:30). Additional sequence at the 5′ ends of these oligonucleotidesprovide homology for yeast recombination cloning when cotransformed withlinearized plasmid pYT-72Bgl (SEQ ID NO:7). In the resulting vector, theexpression of fusion protein SP-GLA (SEQ ID NO:36) is driven by the ADH2promoter. A fusion construct encoding a fusion of an 83 amino acidMFalpha leader peptide (SEQ ID NO:38) N-terminally fused to GLA (SEQ IDNO:37) was cloned using the same techniques. Recombination cloning andgene expression were performed in S. cerevisiae strain INVScl. Directedevolution techniques generally known by those skilled in the art wereused to generate libraries of gene variants from this plasmid construct(See e.g., U.S. Pat. No. 8,383,346 and WO2010/144103).

A chimeric GLA expression construct encoding a synthetic signal peptidefused to a synthetic gene coding for the mature human GLA codingsequence for secreted expression in transient transfections wasgenerated as follows. Oligonucleotides PLEV113Fw (SEQ ID NO:32) andSPGLARv (SEQ ID NO:33) were used to amplify a fragment coding for asynthetic signal peptide (SEQ ID NO:31) using PCR. A second fragmentcoding for the native human coding sequence for the mature form of GLA(SEQ ID NO:4) was amplified using oligonucleotides SPGLAFw (SEQ IDNO:34) and GLARv (SEQ ID NO:35). Splicing by Overlap Extension PCR wasused to recombine these fragments, and the resulting chimeric fragmentwas ligated into the HindIII/Not I linearized mammalian expressionvector pLEV113. Directed evolution techniques generally known by thoseskilled in the art were used to generate specific gene variants fromthis plasmid construct.

Example 2 High-Throughput Growth and Assays High-Throughput (HTP) Growthof GLA and GLA Variants

Yeast (INVScl) cells transformed with vectors expressing GLA and GLAvariants using the lithium acetate method were selected on SD-Ura agarplates. After 72 h incubation at 30° C. colonies were placed into thewells of Axygen® 1.1 ml 96-well deep well plates filled with 200 μl/wellSD-Ura broth (2 g/L SD-Ura, 6.8 g/L yeast nitrogen base without aminoacids [Sigma Aldrich]), 3.06 g/L sodium dihydrogen phosphate, 0.804 g/Ldisodium hydrogen phosphate, pH 6.0 supplemented with 6% glucose. Thecells were allowed to grow for 20-24 hours in a Kuhner shaker (250 rpm,30° C., and 85% relative humidity). Overnight culture samples (20 μL)were transferred into Corning Costar® 96-well deep plates filled with3804 of SD-ura broth supplemented with 2% glucose. The plates wereincubated for 66-84 h in a Kuhner shaker (250 rpm, 30° C., and 85%relative humidity). The cells were then pelleted (4000 rpm×20 min), andthe supernatants isolated and stored at 4° C. prior to analysis.

HTP-Analysis of Supernatants

GLA variant activity was determined by measuring the hydrolysis of4-methylumbelliferyl α-D-galactopyranoside (MUGal). For this assay, 5-50μL of yeast culture supernatant produced as described above, was mixedwith 0-45 μL of McIlvaine Buffer (McIlvaine, J. Biol. Chem., 49:183-186[1921]), pH 4.8 and 50 μL of 2 mM MUGal in 50 mM citrate, 200 mM KCl, pH4.6 in a 96-well, black, opaque bottom plate. The reactions were mixedbriefly and incubated at 37° C. for 30-180 minutes, prior to quenchingwith 100 μL of 1 M sodium carbonate. Hydrolysis was analyzed using aSpectraMax® M2 microplate reader monitoring fluorescence (Ex. 355 nm,Em. 448 nm).

HTP-Analysis of Supernatants Pretreated with Acid

GLA variants were challenged with acidic buffer to simulate the extremepHs that the variants may encounter in lysosomes. First, 50 μL of yeastculture supernatant and 50 uL of McIlvaine buffer (pH 3.3-4.3) wereadded to the wells of a 96-well round bottom plate. The plates weresealed with a PlateLoc Thermal Microplate Sealer (Agilent) and incubatedat 37° C. for 1-3 h. For the assay, 10-50 μL of acid-pH-challengedsample was mixed with 0-40 μL of McIlvaine buffer pH 4.8, 25 μL of 1 Mcitrate buffer pH 4.3 and 25 μL of 4 mM MUGal in McIlvaine buffer pH4.8. The reactions were mixed briefly and incubated at 37° C. for 30-180minutes, prior to quenching with 100 μL of 1 M sodium carbonate.Hydrolysis was analyzed using a SpectraMax® M2 microplate readermonitoring fluorescence (Ex. 355 nm, Em. 448 nm).

HTP-Analysis of Supernatants Pretreated with Base

GLA variants were challenged with basic (neutral) buffer to simulate thepHs that the variants encounter in the blood following theiradministration to a patient. First, 50 μL of yeast culture supernatantand 50 uL of McIlvaine buffer (pH 7.0-8.2) or 200 mM sodium bicarbonate(pH 9.1-9.7) were added to the wells of a 96-well round bottom plate.The plates were sealed and incubated at 37° C. for 1-18 h. For theassay, 10-50 μL of basic-pH-challenged sample was mixed with 0-40 μL ofMcIlvaine buffer pH 4.8, 25 μL of 1 M citrate buffer pH 4.3 and 25 μL of4 mM MUGal in McIlvaine buffer pH 4.8. The reactions were mixed brieflyand incubated at 37° C. for 30-180 minutes, prior to quenching with 100μL of 1 M sodium carbonate. Hydrolysis was analyzed using a SpectraMax®M2 microplate reader monitoring fluorescence (Ex. 355 nm, Em. 448 nm).

HTP-Analysis of Supernatants Pretreated with Bovine Serum

GLA variants were challenged with bovine serum to simulate theconditions the variants encounter following infusion into a patient.First, 20 μL of yeast culture supernatant and 80 μL of bovine serum wereadded to the wells of a 96-well round bottom plate. The plates weresealed and incubated at 37° C. for 1 h. For the assay, 50 μL ofserum-challenged sample was mixed with 25 μL of 1 M citrate buffer pH4.3 and 25 μL of 4 mM MUGal in McIlvaine buffer pH 4.8. The reactionswere mixed briefly and incubated at 37° C. for 180 minutes, prior toquenching with 100 μL of 1 M sodium carbonate. Hydrolysis was analyzedusing a SpectraMax® M2 microplate reader monitoring fluorescence (Ex.355 nm, Em. 448 nm).

TABLE 2.1 Relative Activity of GLA Variants After No Challenge (NC) orChallenge at the Indicated pH^(1,2) SEQ Variant pH pH Amino AcidDifferences Relative ID # NC 4.3 7.0 to SEQ ID NO: 5 NO: 1 + + + A337S47 2 + + + E43D 48 3 + + + E43D/E48D 49 4 + +++ +E43D/E48D/I208V/N247D/Q299R/ 50 Q302K/R373K/I376V 5 ++ ++ ++E43D/E48D/I208V/R373K 51 6 + +++ + E43D/E48D/I208V/R373K/I376V 52 7 +++ + E43D/E48D/N247D/Q299R/ 53 Q302K/R373K/I376V 8 ++ +++ +++E43D/E48D/N247D/Q302K/R373K 54 9 + +++ + E43D/E48D/Q302K/R373K/I376V 5510 ++ +++ ++ E43D/I208V/N247D 56 11 + +++ ++ E43D/I208V/N247D/ 57Q299R/R373K/I376V 12 + ++ + E43D/I208V/Q299R/R373K/I376V 58 13 ++ +++ ++E43D/N247D/R373K/I376V 59 14 + +++ ++ E43D/R373K/I376V 60 15 + + +E48D/I208V/Q299R/Q302K/R373K 61 16 + ++ + E48D/R373K/I376V 62 17 + + +E48G/R373K 63 18 + + ++ F217S 64 19 + ++ + I208V/N247D/Q299R/ 65Q302K/R373K/I376V 20 + +++ ++ I208V/N247D/Q299R/R373K/I376V 66 21 + +++++ I208V/N247D/R373K/I376V 67 22 + + + I208V/Q299R/I376V 68 23 + +++ ++I208V/Q302K/R373K/I376V 69 24 + ++ + I376V 70 25 + + + K36Q 71 26 + + +P179S/R373K 72 27 + + + Q299R/M322V/R373K 73 28 + + + Q299R/Q302K/R373K74 29 + + + Q299R/Q302K/R373K/I376V 75 30 + ++ + Q302K/I376V 76 31 + + +R373K 77 32 + ++ + R373K/I376V 78 ¹Relative activity was calculated asactivity of the variant/activity of WT GLA (SEQ ID NO: 5 (encoded by SEQID NO: 3). ²+ = 0.5 to 1.5 relative activity over WT GLA (SEQ ID NO: 5);++ = >1.5 to 2.5 relative activity over WT GLA (SEQ ID NO: 5); and +++= >2.5 relative activity over WT GLA (SEQ ID NO: 5).

TABLE 2.2 Relative Activity of GLA Variants After No Challenge (NC) orChallenge at the Indicated pH1,2,3 SEQ Variant pH pH Amino AcidDifferences ID # NC 4.2 7.1 Relative to SEQ ID NO: 5 NO: 33 + + +A199H/E367S 79 34 + ++ ++ A337P 80 35 ++ ++ ++ A339S 81 36 + ++ ++ A350G82 37 + + + D105A 83 38 − + − D105S 84 39 + ++ ++D124N/E147G/N161K/R162Q/ 85 T163V/R165A/I167S/V168I/Y169V/S170-/M177S/F217E 40 ++ ++ ++ D396R 86 41 + + + D396T 87 42+++ +++ +++ E367N 88 43 + + + E367T 89 44 + ++ + E387K 90 45 ++ ++ ++E387Q 91 46 + +++ + E387R 92 47 + ++ + E387T 93 48 + + + E40D 9449 + + + F180R 95 50 ++ ++ ++ F180S 96 51 ++ ++ + F198S 97 52 ++ + ++F217D 98 53 + ++ ++ F217R 99 54 + + + F352I 100 55 ++ +++ ++ F352V/F365I101 56 ++ ++ ++ F365I 102 57 ++ ++ ++ F365K 103 58 ++ ++ ++ F365L 10459 + + + F365R 105 60 + + + F365T 106 61 ++ ++ ++ F365V 107 62 ++ + ++G303Q/R373V 108 63 + + ++ H155A 109 64 + + ++ H155L 110 65 + + + H155R111 66 + + + H155T 112 67 ++ + ++ H375E 113 68 + ++ + H84S 114 69 + + +I102L 115 70 + + + I102L/L394V 116 71 ++ + ++ I123T/T369N 117 72 + + +I167V 118 73 + +++ ++ K206A 10 74 + +++ ++ K206M 119 75 + +++ ++ K206Q120 76 + +++ ++ K206R 24 77 + ++ + K206T/V359S 121 78 ++ ++ ++ K343D 12279 ++ ++ ++ K343G 123 80 + + + K362Q 124 81 + + + K362R 125 82 + + +K36D 126 83 + ++ + K36E 127 84 ++ ++ ++ K395* 128 85 + + + K395G 129 86++ ++ ++ K395P 130 87 + ++ + K395R 131 88 + ++ + K395S 132 89 ++ + +K395T 133 90 + + + K96I 134 91 + + ++ K96L 135 92 + + + K96R 136 93 +++++ + K96R/L397V 137 94 + + + L100F 138 95 + + + L158A 139 96 + + +L158I 140 97 + + + L158M 141 98 + + + L158R 142 99 + + + L23M 143100 + + + L23T 144 101 +++ +++ +++ L316D 145 102 +++ +++ +++ L316E 146103 ++ ++ ++ L384F 147 104 ++ ++ ++ L386V 148 105 +++ ++ ++ L394A 149106 ++ ++ +++ L394R 150 107 +++ +++ +++ L394S 151 108 +++ +++ +++ L394T152 109 ++ ++ +++ L397* 153 110 +++ ++ +++ L397D 154 111 ++ ++ ++ L397H155 112 + ++ + L397I 156 113 ++ + +++ L397Q 157 114 ++ ++ ++ L397R 158115 ++ ++ +++ L397T 159 116 ++ ++ ++ L398E 160 117 ++ ++ ++ L398G 161118 ++ ++ ++ L398N 162 119 ++ ++ ++ L398Q 163 120 ++ ++ ++ L398R 164121 + ++ ++ L44R/L384F 165 122 ++ ++ ++ L44T 166 123 − + − M20D/Q302K167 124 ++ ++ + M253F 168 125 + + + M322I 169 126 +++ +++ +++ M390D 170127 ++ ++ ++ M390R 171 128 + + + M390T 172 129 + ++ ++ M392G 173 130 +++ + M392P 174 131 ++ + ++ M392S 175 132 + + + M39Y 176 133 + + + N388R177 134 + + + N91Q 178 135 ++ +++ ++ Q190S/T369D 179 136 + + + Q249A 180137 + + + Q302A 181 138 ++ ++ ++ Q385H 182 139 + + + Q385I 183 140 ++ ++++ Q385L 184 141 + + + Q391G 185 142 + + + Q80A 186 143 + + + Q80H 187144 + ++ + Q80V 188 145 + + + Q88A 189 146 + + + Q88F 190 147 ++ ++ ++Q88H 191 148 + ++ + Q88R 192 149 ++ + ++ Q88S 193 150 + + + R162H 194151 + + + R162S 195 152 ++ ++ ++ R221K/A350G 196 153 + + ++ R221T 197154 ++ + ++ R301I/K362T 198 155 + + + R301L 199 156 ++ + ++ R371S 200157 ++ + ++ R371V 201 158 ++ + ++ R87K 202 159 + + + R87P/L398R 203160 + ++ + S166A 204 161 + + + S166H 205 162 + + + S166K 206 163 + + +S31D 207 164 + − − S34D/M392P 208 165 + − − S34G 209 166 ++ + +S34H/M390R 210 167 + + + S34R 211 168 ++ ++ ++ S374M 212 169 ++ ++ ++S374T 213 170 ++ ++ ++ S393E 214 171 ++ ++ ++ S393G 215 172 + + + S393H216 173 ++ ++ ++ S393P 217 174 + + + S47I 218 175 + ++ + S47R 219176 + + + S47T 220 177 + ++ + S95D 221 178 ++ +++ ++ S95E 222 179 + + +S95Q 223 180 ++ ++ +++ T369D 224 181 + + + T369S 225 182 ++ + + T389S226 183 + + + V133I 227 184 ++ + + V168A 228 185 + ++ + V168L 229 186 ++++ +++ V345N 230 187 + + + V345Y 231 188 + + + V359E 232 189 + + + V93I233 190 ++ + ++ W178H 234 191 + ++ + W178S 235 ¹Relative activity wascalculated as activity of the variant/activity of WT GLA (SEQ ID NO: 5(encoded by SEQ ID NO: 3). ²Variant # 73 (Rd2BB) has the polynucleotidesequence of SEQ ID NO: 8 and polypeptide sequence of SEQ ID NO: 10. ³− =<0.5 relative activity to WT GLA (SEQ ID NO: 5); + = 0.5 to 1.5 relativeactivity over WT GLA (SEQ ID NO: 5); ++ = >1.5 to 2.5 relative activityover WT GLA (SEQ ID NO: 5); and +++ = >2.5 relative activity over WT GLA(SEQ ID NO: 5).

TABLE 2.3 Relative Activity of GLA Variants After No Challenge (NC) orChallenge at the Indicated pH Variant pH pH Amino Acid Differences SEQID # NC 4.2 7.6 Relative to SEQ ID NO: 10 NO: 192 + + + A206E 236193 + + + A206G 237 194 ++ ++ ++ A206R 238 195 + + + A206S 239 196 + + +A350G 240 197 ++ ++ ++ A350G/K362Q/T369A 241 198 ++ ++ +++ A350G/T369D242 199 ++ ++ ++ A350G/T369S 243 200 + + + C143A 244 201 + + + C143T 245202 + + + C59A 246 203 ++ ++ +++ E367A/T369D 247 204 + + + E367D 248 205++ ++ ++ E367D/T369D 21 206 +++ +++ +++ E367N 18 207 ++ +++ +++E367N/R373K 249 208 ++ +++ +++ E367N/R373K/I376V 250 209 + + +E367P/T369D 251 210 ++ ++ ++ F365L/E367N 252 211 ++ ++ ++F365L/E367N/I376V 253 212 ++ ++ ++ F365L/E367N/R373K/I376V 254 213 + − −H15Q/ 255 214 +++ +++ +++ K343D/F365L/E367N 256 215 + + + K343G 257 216++ +++ +++ K343G/F365L/E367N/R373K 258 217 ++ ++ ++ L316D 259 218 ++++++ +++ M322I/E367N/R373K 13 219 + + + M322I/R373K 260 220 + + ++M322V/R373K/I376V 261 221 + + + M390I 262 222 ++ ++ + P228Q/T369D 263223 + ++ ++ Q302K/A337P/A350G/K362Q 264 224 ++ +++ +++ Q302K/M322V/E367N265 225 + + + R165S 266 226 + + ++ R221T/F365L 267 227 − − − R325H 268228 + + + R373K 269 229 + − + R373K/I376V 270 230 + − + S374R 271 231 ++++ ++ T369D 272 232 ++ ++ ++ T369S 273 1. Relative activity wascalculated as activity of the variant/activity of Rd2BB (SEQ ID NO: 10)2. Variant # 218 (Rd3BB) has the polynucleotide sequence of SEQ ID NO:11 and polypeptide sequence of SEQ ID NO: 13. 3. − = <0.5 relativeactivity to Rd2BB (SEQ ID NO: 10); + = 0.5 to 1.5 relative activity overRd2BB (SEQ ID NO: 10); ++ = >1.5 to 2.5 relative activity over Rd2BB(SEQ ID NO: 10); and +++ = >2.5 relative activity over Rd2BB (SEQ ID NO:10).

TABLE 2.4 Relative Activity of GLA Variants After No Challenge (NC) orChallenge at the Indicated pH or Condition^(1,2) Variant pH pH AminoAcid Differences Relative to SEQ ID # NC 4.2 7.6 Serum SEQ ID NO: 5 (WTGLA) NO: 233 +++ +++ +++ +++ K206A/F217R/N247D/L316D/A350G/E367D/T369D274 234 +++ +++ +++ +++ K206A/F217R/N247D/Q302K/A350G/E367D/T369D 275235 +++ +++ +++ +++ K206A/F217R/N247D/Q302K/L316D/A337P/A350G/ 276E367D/T369D 236 +++ ++ +++ ++ K206A/F217R/Q302K/E367D/T369D 277 237 ++++++ +++ +++ K206A/F217R/Q302K/L316D/A337P/A350G/E367D/ 278 T369D 238 +++++ +++ ++ K206A/I208V/M322V/K343G/F365L/R373K/I376V 279 239 +++ +++ +++++ K206A/I208V/R221K/N247D/M322I/K343D/F365L/ 280 R373K/I376V 240− + + + K206A/L269I/P349L/R373K 281 241 ++ +++ +++ ++K206A/N247D/M322V/K343D/R373K/I376V 282 242 +++ +++ +++ +++K206A/N247D/M322V/K343G/F365L/R373K 283 243 +++ +++ +++ +++K206A/N247D/Q302K/A337P/K343G/A350G 284 244 +++ +++ +++ +++K206A/N247D/Q302K/L316D/A350G 285 245 ++ +++ +++ ++K206A/N247D/Q302K/M322V/F365L/R373K/I376V 286 246 +++ +++ +++ +++K206A/Q302K/L316D/A337P 287 247 +++ +++ +++ +++K206A/R221K/N247D/M322V/K343D/R373K 288 248 + + + +K206A/R221T/M322V/K343G/R373K 289 249 ++ ++ +++ ++K206A/R221T/M322V/R373K 290 250 + + + + K96I/K206A/F217R 291 251 ++ +++ + K96I/K206A/F217R/N247D 292 252 +++ +++ +++ +++K96I/K206A/F217R/N247D/A350G/E367D/T369D 293 253 +++ +++ +++ +++K96I/K206A/F217R/N247D/Q302K/L316D/A337P/ 294 E367D/T369D 254 + + + +L100F/K206A 295 255 +++ +++ +++ ++L100F/K206A/I208V/R221K/N247D/Q302K/M322I/ 296 K343D/F365L/I376V 256 +++++ +++ +++ L100F/K206A/I208V/R221K/N247D/Q302K/M322V/ 297K343D/F365L/I376V 257 ++ ++ ++ ++L100F/K206A/I208V/R221T/N247D/K343D/F365L/I376V 298 258 +++ ++ +++ ++L100F/K206A/I208V/R221T/Q302K/M322I/K343D/I376V 299 259 + + + +L100F/K206A/M322V/F365L/R373K/I376V 300 260 + + + +L100F/K206A/N247D/F365L/R373K/I376V 301 261 ++ ++ ++ +L100F/K206A/N247D/M322V/K343D/I376V 302 262 ++ +++ +++ +++L100F/K206A/R221K/N247D/Q302K/M322V/F365L/ 303 R373K/I376V 263 + ++ +++++ L100F/K206A/R221K/N247D/Q302K/M322V/I376V 304 264 ++ ++ +++ ++L100F/K206A/R221K/N247D/Q302K/M322V/K343D/ 305 R373K/I376V 265 + + + +L100F/K206A/R221K/R373K/I376V 306 266 ++ ++ +++ ++L100F/K206A/R221T/M322I/K343E/F365L/R373K 307 267 +++ +++ +++ +++L100F/K206A/R221T/N247D/Q302K/K343D/F365L/ 308 R373K 268 + + + +L100F/K206A/R373K/I376V 309 269 − + + +L37I/K206A/R221K/N247D/M322I/R373K 310 270 + + + +L44R/C143Y/K206A/A337P/A350G 311 271 − + + +L44R/E187G/K206A/A337P/A350G 312 272 + + + + L44R/K206A 313 273 + + + +L44R/K206A/E367D/T369D 314 274 + + + + L44R/K206A/F217R/A350G 315 275 ++++ ++ ++ L44R/K206A/F217R/N247D/A337P 316 276 +++ +++ +++ +++L44R/K206A/F217R/N247D/L316D/A337P/A350G/ 317 E367D/T369D 277 +++ ++++++ +++ L44R/K206A/F217R/N247D/L316D/A337P/E367D/ 318 T369D 278 +++ ++++++ +++ L44R/K206A/F217R/N247D/L316D/A350G/E367D/ 319 T369D 279 ++ ++++++ +++ L44R/K206A/F217R/N247D/Q302K/A350G 320 280 + + + +L44R/K206A/F217R/Q302K/E367D/T369D 321 281 + + + +L44R/K206A/I208V/R221K/M322V/K343D/F365L/ 322 R373K 282 + + + +L44R/K206A/N247D/A337P 323 283 +++ +++ +++ +++L44R/K206A/N247D/Q302K/A337P/A350G/E367D/ 324 T369D 284 +++ +++ +++ +++L44R/K206A/R221T/N247D/M322I/K343D/F365L/ 325 I376V 285 + + ++ +L44R/K96I/K206A 326 286 + + ++ + L44R/K96I/K206A/F217R/N247D 327 287 ++++++ +++ ++ L44R/K96I/K206A/F217R/N247D/Q302K/A337P/ 328 A350G 288 ++++++ +++ +++ L44R/K96I/K206A/F217R/N247D/Q302K/A337P/ 329K343D/A350G/E367D/T369D 289 + ++ ++ ++ L44R/K96I/K206A/F217R/Q302K/A350G330 290 +++ +++ +++ +++ L44R/K96I/K206A/N247D/L316D/A337P/A350G/ 331E367D/T369D 291 + + + + L44R/L100F/K206A/F365L 332 292 +++ ++ +++ ++L44R/L100F/K206A/I208V/Q219H/N247D/Q302K/ 333 M322V/K343D/R373K/I376V293 ++ + ++ + L44R/L100F/K206A/I208V/R221K/N247D/Q302K/ 334M322V/F365L/I376V 294 ++ ++ +++ +L44R/L100F/K206A/I208V/R221T/N247D/M322V/ 335 I376V 295 +++ +++ +++ +++L44R/L100F/K206A/I208V/R221T/N247D/Q302K/ 336M322I/K343D/F365L/R373K/I376V ¹Relative activity was calculated asactivity of the variant/activity of Rd2BB (SEQ ID NO: 10 (encoded by SEQID NO: 8). ²− = <0.5 relative activity to Rd2BB (SEQ ID NO: 10); + = 0.5to 1.5 relative activity over Rd2BB (SEQ ID NO: 10); ++ = >1.5 to 2.5relative activity over Rd2BB (SEQ ID NO: 10); and +++ = >2.5 relativeactivity over Rd2BB (SEQ ID NO: 10).

TABLE 2.5 Relative Activity of GLA Variants After No Challenge (NC) orChallenge at the Indicated pH or Condition^(1,2,3) Variant pH Amino AcidDifferences Relative to SEQ ID # NC pH 4.0 8.2 Serum SEQ ID NO: 5 (WTGLA) NO: 296 + − − + A66T/K206A/F217R/L316D/M322I/A337P/ 337K343G/A350G/E367N/R373K 297 − − − − K206A/F217R/G230V/N247D/Q302K/M322I/338 E3673N/T369S/R373K 298 ++ +++ +++ +++K206A/F217R/N247D/L316D/M322I/A337P/ 339 A350G/K362Q/E367N/R373K 299 + −− − K206A/F217R/N247D/Q249H/Q302K/M322I/ 340K343G/A350G/E367T/R373K/L397F 300 + ++ ++ ++K206A/I208V/R221T/N247D/M322V/K343G/ 341 E367N/R373K 301 + + + −K206A/M322I/E367N/R373K 342 302 + + + + K206A/M322V/K343G/E367N/R373K343 303 ++ ++ ++ + K206A/N247D/M322I/A337E/K343D/F365L/ 344E367N/R373K/I376V 304 ++ ++ ++ ++ K206A/Q302K/L316D/M322I/A337P/A350G/345 K362Q/E367N/T369S/R373K 305 + + + ++K206A/Q302K/L316D/M322I/A337P/K343D/ 346 E367N/T369S/R373K 306 + ++ ++++ K206A/R221K/N247D/Q302K/M322I/E367N/ 347 R373K 307 + + + +K206A/R221K/Q302K/M322I/K343G/E367N/ 348 R373K/I376V 308 − − − +K96I/K206A/F217R/M322I/E367N/T369S/ 349 R373K 309 + ++ +++ ++K96I/K206A/F217R/N247D/Q302K/M322I/ 350 A337P/K343G/A350G/E367N/R373K310 − + + + K96I/K206A/N247D/M322I/A350G/E367N/ 351 T369S/R373K 311 + +++ + K96I/K206A/N247D/Q302K/L316D/M322I/ 352A337P/A350G/E367N/T369S/R373K 312 ++ +++ +++ +++K96I/K206A/N247D/Q302K/L316D/M322I/ 353A337P/A350G/K362Q/E367N/T369S/R373K 313 + + − +L100F/A125S/K206A/I208V/R221K/Q302K/ 354 M322I/K343G/E367N/R373K 314 +++ ++ + L100F/K206A/I208V/N247D/Q302K/M322V/ 355 K343D/E367N/R373K/I376V315 + + + + L100F/K206A/I208V/Q302K/M322V/F365L/ 356 E367N/R373K/I376V316 + + + + L100F/K206A/I208V/R221K/M322V/K343D/ 357 E367N/R373K 317 + +− − L100F/K206A/I208V/R221K/M322V/K343D 358 F365L/E367N/R373K 318 + +++ + L100F/K206A/I208V/R221T/M322V/E367N/ 359 R373K/I376V 319 + + + +L100F/K206A/M322I/E367N/R373K/I376V 360 320 + + + +L100F/K206A/N247D/Q302K/M322I/E367N/R373K 361 321 ++ ++ +++ +L100F/K206A/R221K/N247D/M322I/K343G/ 362 E367N/R373K 322 + + ++ +L100F/K206A/R221T/Q302K/M322I/K343D/ 363 E367N/R373K 323 − − − +L100F/L1601/K206A/R221K/M322V/E367N/R373K 364 324 − − − −L23S/K206A/M322I/E367N/R373K 365 325 ++ +++ +++ +++L44R/K206A/F217R/N247D/L316D/M322I/ 366 A337P/K343G/K362Q/E367N/R373K326 ++ +++ +++ +++ L44R/K206A/F217R/N247D/Q302K/L316D/ 367M322I/A337P/K362Q/E367N/R373K 327 ++ +++ +++ +++L44R/K206A/F217R/N247D/Q302K/L316D/ 368M322I/K343D/A350G/K362Q/E367N/R373K 328 + + + +L44R/K206A/F217R/Q302K/M322I/A337P/ 369 A350G/E367N/T369S/R373K 329 + ++++ ++ L44R/K206A/I208V/N247D/Q302K/M322I/ 370 K343D/E367N/R373K 330 ++++ + + L44R/K206A/I208V/R221K/M322I/K343D/ 371 E367N/R373K 331 + ++ ++ +L44R/K206A/I208V/R221K/N247D/Q302K/ 372 M322I/K343D/E367N/R373K/I376V332 + + + + L44R/K206A/I208V/R221T/Q302K/M322I/ 373K343G/F365L/E367N/R373K/I376V 333 ++ ++ ++ +L44R/K206A/L316D/M322I/A337P/A350G/ 374 E367N/T369S/R373K 334 + ++ +++++ L44R/K206A/N247D/L316D/M322I/A350G/ 375 K362Q/E367N/T369S/R373K 335++ +++ +++ +++ L44R/K206A/N247D/Q302K/L316D/M322I/ 376A337P/K343G/A350G/K362Q/E367N/ T369S/R373K 336 + + + −L44R/K206A/N247D/Q302K/M322I/A350G/ 377 E367N/T369S/R373K 337 + ++ ++ ++L44R/K206A/N247D/Q302K/M322I/K343D/ 378 E367N/R373K 338 ++ +++ +++ +++L44R/K96I/K206A/F217R/N247D/L316D/ 379M322I/A337P/A350G/K362Q/E367N/R373K 339 + + ++ +L44R/K96I/K206A/F217R/N247D/M322I/ 380 A350G/K362Q/E367N/R373K340 + + + + L44R/K96I/K206A/F217R/N247D/M322I/ 381A350G/K362Q/E367N/T369S/R373K 341 − + + +L44R/K96I/K206A/F217R/N247D/M322I/ 382 E367N/T369S/R373K 342 ++ +++ ++++++ L44R/K96I/K206A/F217R/N247D/Q302K/ 383 L316D/M322I/A337P/E367N/R373K343 + + + + L44R/K96I/K206A/F217R/N247D/Q302K/ 384M322I/E367N/T369S/R373K 344 + + + ++ L44R/K96I/K206A/F217R/N247D/Q302K/385 M322I/K362Q/E367N/R373K 345 + ++ +++ +L44R/K96I/K206A/F217R/Q219P/N247D/ 386M253K/S266F/D284E/Q290P/L293F/Q302K/ V308G/S314F/M322I/A337P/K343E/E367N/R373K 346 + + ++ + L44R/K96I/K206A/F217R/Q302K/M322I/ 387A350G/K362Q/E367N/T369S/R373K 347 − − − −L44R/K96I/K206A/M322I/A337P/E367N/ 388 T369S/R373K 348 + + + +L44R/L100F/K206A/I208V/R221K/M322I/ 389 K343G/F365L/E367N/R373K 349 + +++ + L44R/L100F/K206A/I208V/R221T/N247D/ 390 M322I/F365L/E367N/R373K350 + ++ +++ + L44R/L100F/K206A/I208V/R221T/N247D/ 391M322V/E367N/R373K/I376V 351 + + ++ + L44R/L100F/K206A/I208V/R221T/Q302K/392 M322I/E367N/R373K/I376V 352 + + + +L44R/L100F/K206A/Q302K/M322I/E367N/ 393 R373K/I376V 353 − − − +L44R/L100F/K206A/R221K/M322I/F365L/ 394 E367N/R373K/I376V 354 − + + +L44R/L100F/K206A/R221T/M322I/F365L/ 395 E367N/R373K 355 + ++ ++ +L44R/L100F/K206A/R221T/N247D/M322I/ 396 K343D/E367N/R373K/I376V 356 + +++ + L44R/L100F/K206A/R221T/N247D/Q302K/ 397 M322I/E367N/R373K 357 + +++++ + L44R/L100F/K206A/R221T/N247D/Q302K/ 398 M322V/E367N/R373K/I376V358 + + ++ + L44R/L100F/K206A/R221T/Q302K/M322I/ 399 E367N/R373K 359 + ++++ + L44R/L100F/Q181L/K206A/R221T/N247D/ 400Q302K/M322V/E367N/R373K/I376V ¹Relative activity was calculated asactivity of the variant/activity of Rd3BB (SEQ ID NO: 13 (encoded by SEQID NO: 11). ²Variant #326 (Rd4BB) has the polynucleotide sequence of SEQID NO: 14 and polypeptide sequence of SEQ ID NO: 15. ³− = <0.5 relativeactivity to Rd3BB (SEQ ID NO: 13); + = 0.5 to 1.5 relative activity overRd3BB (SEQ ID NO: 13); ++ = >1.5 to 2.5 relative activity over Rd3BB(SEQ ID NO: 13); and +++ = >2.5 relative activity over Rd3BB (SEQ ID NO:13).

TABLE 2.6 Relative Activity of GLA Variants After No Challenge (NC) orChallenge at the Indicated pH or Condition^(1,2,3,4) Var- SEQ iant pH pHAmino acid differences relative ID # NC 3.7 9.65 to SEQ ID NO: 5 (WTGLA) NO: 360 + + + L44E/K206A/F217R/N247D/ 401 Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 361 + + + L44R/S47R/K206A/F217R/ 402N247D/Q302K/L316D/ M322I/A337P/K362Q/E367N/R373K 362 + + +L44C/K206A/F217R/N247D/ 403 Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K363 + + + L44R/S47D/K206A/F217R/ 404 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 364 + + − M39H/L44R/K206A/F217R/ 405N247D/Q302K/L316D/ M322I/A337P/K362Q/E367N/R373K 365 + + +L44R/S47N/K206A/F217R/ 406 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 366 + + + L44R/S47V/K206A/F217R// 407N247D/Q302K/L316D M322I/A337P/K362Q/E367N/R373K 367 + + −M39R/L44R/K206A/F217R/ 408 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 368 + + + L44A/K206A/F217R/N247D/ 409Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K 369 + + −L44S/K206A/F217R/N247D/ 410 Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K370 + ++ + L44Q/K206A/F217R/N247D/ 411 Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 371 + − + L44W/K206A/F217R/N247D/ 412Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K 372 + − +L44V/K206A/F217R/N247D/ 413 Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K373 − − − M41R/L44R/K206A/F217R/ 414 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 374 + + + L44M/K206A/F217R/N247D/ 415Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K 375 + + +L44R/S47I/K206A/F217R/ 416 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 376 − − − M41P/L44R/K206A/F217R/ 417N247D/Q302K/L316D/ M322I/A337P/K362Q/E367N/R373K 377 + ++ −M39T/L44R/K206A/F217 R/ 418 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 378 + − + L44T/K206A/F217R/N247D/ 419Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K 379 + ++ +L44R/S47T/K206A/F217R/ 420 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 380 + + − L44R/Y92K/K206A/F217R/ 421N247D/Q302K/L316D/ M322I/A337P/K362Q/E367N/R373K 381 + + −L44R/Y92S/K206A/F217R/ 422 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 382 − − − L44R/H94N/K206A/F217R/ 423N247D/Q302K/L316D/ M322I/A337P/K362Q/E367N/R373K 383 + − −L44R/Y92C/K206A/F217R/ 424 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 384 + + − L44R/Y92V/K206A/F217R/ 425N247D/Q302K/L316D/ M322I/A337P/K362Q/E367N/R373K 385 + + −L44R/Y92A/K206A/F217R/ 426 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 386 − + − L44R/H94R/K206A/F217R/ 427N247D/Q302K/L316D/ M322I/A337P/K362Q/E367N/R373K 387 + + −L44R/V93T/K206A/F217R/ 428 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 388 + − + L44R/V93L/K206A/F217R/ 429N247D/Q302K/L316D/ M322I/A337P/K362Q/E367N/R373K 389 + + −L44R/V93S/K206A/F217R/ 430 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 390 + + − L44R/Y92Q/K206A/F217R/ 431N247D/Q302K/L316D/ M322I/A337P/K362Q/E367N/R373K 391 + + −L44R/Y92W/K206A/F217R/ 432 N247D/Q302K/L316D/ M322I/A337P/K362Q/E367N/R373K/L397S 392 + + − L44R/Y92T/K206A/F217R/ 433N247D/Q302K/L316D/ M322I/A337P/K362Q/E367N/R373K 393 + − −L44R/Y92G/K206A/F217R/ 434 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 394 + + − L44R/Y92R/K206A/F217R/ 435N247D/Q302K/L316D/ M322I/A337P/K362Q/E367N/R373K 395 + + +L44R/Y92H/K206A/F217R/ 40 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 396 + + + L44R/L158M/K206A/F217R/ 437N247D/Q302K/L316D/ M322I/A337P/K362Q/E367N/R373K 397 + + +L44R/L158R/K206A/ 438 F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 398 + ++ − L44R/A159S/K206A/F217R/ 439N247D/Q302K/L316D/ M322I/A337P/K362Q/E367N/R373K 399 + + +L44R/R165K/K206A/F217R/ 440 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 400 + + − L44R/L158C/K206A/F217R/ 441N247D/Q302K/L316D/ M322I/A337P/K362Q/E367N/R373K 401 + + −L44R/T163S/K206A/F217R/ 442 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 402 + ++ + L44R/S166P/K206A/F217R/ 42N247D/Q302K/L316D/ M322I/A337P/K362Q/E367N/R373K 403 + + +L44R/S166G/K206A/F217R/ 444 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 404 + + − L44R/S166F/K206A/F217R/ 445N247D/Q302K/L316D/ M322I/A337P/K362Q/E367N/R373K 405 + ++ +L44R/L158E/K206A/F217R/ 446 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 406 + + + L44R/R162K/K206A/ 447F217R/N247D/Q302K/L316D/ M322I/A337P/K362Q/E367N/R373K 407 + + −L44R/L158H/K206A/F217R/ 448 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 408 + + + L44R/S166R/K206A/F217R/ 449N247D/Q302K/L316D/ M322I/A337P/K362Q/E367N/R373K 409 + + −L44R/R165H/K206A/F217R/ 450 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 410 + + − L44R/R162H/K206A/F217R/ 451N247D/Q302K/L316D/ M322I/A337P/K362Q/E367N/R373K 411 + + +L44R/S166A/K206A/F217R/ 452 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 412 + ++ + L44R/S166H/K206A/F217R/ 453N247D/Q302K/L316D/ M322I/A337P/K362Q/E367N/R373K 413 − − −L44R/T163*/K206A/F217R/ 454 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 414 + + + L44R/L158Q/K206A/F217R/ 455N247D/Q302K/L316D/ M322I/A337P/K362Q/E367N/R373K 415 + + +L44R/S166D/K206A/F217R/ 456 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 416 + + − L44R/R162G/K206A/F217R/ 457N247D/Q302K/L316D/ M322I/A337P/K362Q/E367N/R373K 417 + + −L44R/R162S/K206A/F217R/ 458 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 418 + + − L44R/N161E/K206A/F217R/ 459N247D/Q302K/L316D/ M322I/A337P/K362Q/E367N/R373K 419 + + +L44R/S166E/K206A/F217R/ 460 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 420 + ++ − L44R/S166T/K206A/F217R/ 461N247D/Q302K/L316D/ M322I/A337P/K362Q/E367N/R373K 421 + + −L44R/R162Q/K206A/F217R/ 462 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 422 + + − L44R/L158G/K206A/F217R/ 463N247D/Q302K/L316D/ M322I/A337P/K362Q/E367N/R373K 423 + + +L44R/R162A/K206A/F217R/ 464 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 424 + + − L44R/K206A/F217R/N247D/ 465L255E/Q302K/L316D/ M322I/A337P/K362Q/E367N/R373K 425 + − +L44R/K206A/F217R/N247D/ 466 H271E/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 426 + − − L44R/K206A/F217R/N247D/ 467M259E/Q302K/L316D/ M322I/A337P/K362Q/E367N/R373K 427 + − −L44R/K206A/F217R/N247D/ 468 L263G/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 428 + + − L44R/K206A/F217R/N247D/ 469M259S/Q302K/L316D/ M322I/A337P/K362Q/E367N/R373K 429 + + −L44R/K206A/F217R/N247D/ 470 L255C/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 430 + − + L44R/K206A/F217R/N247D/ 471H271T/Q302K/L316D/ M322I/A337P/K362Q/E367N/R373K 431 + − −L44R/K206A/F217R/N247D/ 472 R270G/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 432 + − + L44R/K206A/F217R/N247D/ 473L255V/Q302K/L316D/ M322I/A337P/K362Q/E367N/R373K 433 + + +L44R/K206A/F217R/N247D/ 474 H271Q/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 434 + − − L44R/K206A/F217R/N247D/ 475R270D/Q302K/L316D/ M322I/A337P/K362Q/E367N/R373K 435 + ++ −L44R/K206A/F217R/N247D/ 476 I258L/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 436 + − − L44R/K206A/F217R/N247D/ 477H271G/Q302K/L316D/ M322I/A337P/K362Q/E367N/R373K 437 + + −L44R/K206A/F217R/N247D/ 478 L263E/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 438 − − − L44R/K206A/F217R/N247D/ 479L255*/Q302K/L316D/ M322I/A337P/K362Q/E367N/R373K 439 + + +L44R/K206A/F217R/N247D/ 480 H271A/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 440 + + − L44R/K206A/F217R/N247D/ 481L263C/Q302K/L316D/ M322I/A337P/K362Q/E367N/R373K 441 + − +L44R/K206A/F217R/N247D/ 482 H271V/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 442 + + − L44R/K206A/F217R/N247D/ 483L255A/Q302K/L316D/ M322I/A337P/K362Q/E367N/R373K 443 + +++ −L44R/K206A/F217R/N247D/ 484 L255S/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 444 + − + L44R/K206A/F217R/N247D/ 485M259W/Q302K/L316 D/M322I/A337P/K362Q/ E367N/R373K 445 + + −L44R/K206A/F217R/N247D/ 486 L263F/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 446 + − − L44R/K206A/F217R/N247D/ 487M259A/Q302K/L316D/ M322I/A337P/K362Q/E367N/R373K 447 + − −L44R/K206A/F217R/N247D/ 488 L263W/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 448 + − − L44R/K206A/F217R/N247D/ 489R270Q/Q302K/L316D/ M322I/A337P/K362Q/E367N/R373K 449 + ++ −L44R/K206A/F217R/N247D/ 490 L255T/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 450 + ++ − L44R/K206A/F217R/N247D/ 491I258M/Q302K/L316D/ M322I/A337P/K362Q/E367N/R373K 451 + ++ −L44R/K206A/F217R/N247D/ 492 M259V/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 452 + ++ + L44R/K206A/F217R/N247D/ 493H271R/Q302K/L316D/ M322I/A337P/K362Q/E367N/R373K 453 + − −L44R/K206A/F217R/N247D/ 494 R270L/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 454 + ++ + L44R/K206A/F217R/N247D/ 495Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K/M390P 455 + + +L44R/K206A/F217R/N247D/ 496 Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392D 456 + + + L44R/K206A/F217R/N247D/ 497Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K/T389M 457 + + +L44R/K206A/F217R/N247D/ 498 Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392A 458 + + + L44R/K206A/F217R/N247D/ 499Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K/M390* 459 + ++ +L44R/K206A/F217R/N247D/ 500 Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M390H 460 + + + L44R/K206A/F217R/N247D/ 501Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K/L386T 461 + + +L44R/K206A/F217R/N247D/ 502 Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392Q 462 + + + L44R/K206A/F217R/N247D/ 503Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K/Q385L 463 + + +L44R/K206A/F217R/N247D/ 504 Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M390T 464 + + + L44R/K206A/F217R/N247D/ 505Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K/M392* 465 + + +L44R/K206A/F217R/N247D/ 506 Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M390Q 466 + + + L44R/K206A/F217R/N247D/ 507Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K/M392E 467 + + +L44R/K206A/F217R/N247D/ 508 Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/T389S 468 + + + L44R/K206A/F217R/N247D/ 509Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K/T389Q 469 + + +L44R/K206A/F217R/N247D/ 510 Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/Q385I 470 + ++ + L44R/K206A/F217R/N247D/ 511Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K/M392R 471 + + +L44R/K206A/F217R/N247D/ 512 Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/T389W 472 + + + L44R/K206A/F217R/N247D/ 513Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K/M392K 473 + + +L44R/K206A/F217R/N247D/ 514 Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392L 474 + ++ + L44R/K206A/F217R/N247D/ 515Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K/L386F 475 + + +L44R/K206A/F217R/N247D/ 516 Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/T389D 476 + + + L44R/K206A/F217R/N247D/ 517Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K/M390E 477 + − +L44R/K206A/F217R/N247D/ 518 Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/L384W 478 + ++ + L44R/K206A/F217R/N247D/ 519Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K/M392S 479 + + +L44R/K206A/F217R/N247D/ 520 Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392F 480 + + + L44R/K206A/F217R/N247D/ 521Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K/M390R 481 + + +L44R/K206A/F217R/N247D/ 522 Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M390G 482 + + + L44R/K206A/F217R/N247D/ 523Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K/Q385G 483 + + +L44R/K206A/F217R/N247D/ 524 Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392C 484 + + + L44R/K206A/F217R/N247D/ 525Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K/M392V 485 + + +L44R/K206A/F217R/N247D/ 526 Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392W 486 + + + L44R/K206A/F217R/N247D/ 527Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K/M390C 487 + + +L44R/K206A/F217R/N247D/ 528 Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/T389G 488 + + + L44R/K206A/F217R/N247D/ 529Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K/T389N 489 + + +L44R/K206A/F217R/N247D/ 530 Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/T389I 490 + + + L44R/K206A/F217R/N247D/ 531Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K/M390D 491 + + +L44R/K206A/F217R/N247D/ 532 Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M390W 492 + + + L44R/K206A/F217R/N247D/ 533Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K/T389C 493 + + +L44R/K206A/F217R/N247D/ 534 Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392P 494 + + + L44R/K206A/F217R/N247D/ 535Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K/M390F 495 + + +L44R/K206A/F217R/N247D/ 536 Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/T389P 496 + + + L44R/K206A/F217R/N247D/ 537Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K/M390V 497 + ++ +L44R/K206A/F217R/N247D/ 538 Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M390K 498 + + + L44R/K206A/F217R/N247D/ 539Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K/M392I 499 + + +L44R/K206A/F217R/N247D/ 540 Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/T389L 500 + + + L44R/K206A/F217R/N247D/ 541Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K/M390A 501 + ++ +L44R/K206A/F217R/N247D/ 542 Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392G 502 − + − L44R/K206A/F217R/N247D/ 543Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K/L386S 503 + + +L44R/K206A/F217R/N247D/ 544 Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/Q385C 504 + + + L44R/K206A/F217R/N247D/ 545Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K/M390S 505 + + +L44R/K206A/F217R/N247D/ 546 Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392N 506 + + − L44R/K206A/F217R/N247D/ 547Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K/Q385W 507 + ++ +L44R/K206A/F217R/N247D/ 548 Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392T 508 − − − L44R/K206A/F217R/N247D/ 549Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K/L384A 509 + + +L44R/K206A/F217R/N247D/ 550 Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/Q385T 510 + − + L44R/A199G/K206A/F217R/ 551N247D/Q302K/L316D/ M322I/A337P/K362Q/E367N/ R373K/M392R 511 + + +L44R/K206A/F217R/N247D/ 552 Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/L397* 512 + + + L44R/K206A/F217R/N247D/ 553Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K/K395* 513 + + +L44R/K206A/F217R/N247D/ 554 Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/D396* 514 + + + L44R/K206A/F217R/N247D/ 555Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K/S393* 515 + + +L44R/K206A/F217R/N247D/ 556 Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/L394* ¹Relative activity was calculated asactivity of the variant/activity of Rd4BB (SEQ ID NO: 15 (encoded by SEQID NO: 14). ²Variant # 395 (Rd5BB) has the polynucleotide sequence ofSEQ ID NO: 39 and polypeptide sequence of SEQ ID NO: 40. ³Variant # 402(Rd6BB) has the polynucleotide sequence of SEQ ID NO: 41 and polypeptidesequence of SEQ ID NO: 42 ⁴− = <0.5 relative activity to Rd4BB (SEQ IDNO: 15); + = 0.5 to 1.5 relative activity over Rd4BB (SEQ ID NO: 15); ++= >1.5 to 2.5 relative activity over Rd4BB (SEQ ID NO: 15); and +++= >2.5 relative activity over Rd4BB (SEQ ID NO: 15).

TABLE 2.7 Relative Activity of GLA Variants After No Challenge (NC) orChallenge at the Indicated pH or Condition Var- SEQ iant PH PH Aminoacid differences relative to ID # NC 3.7 9.7 SEQ ID NO: 5 (WT GLA) NO:516 + +++ + D2E/L44R/Y92H/K206A/F217R/ 557 N247D/Q302K/L316D/M322I/Q326G/A337P/K362Q/E367N/R373K 517 + + + D2Q/L44R/Y92H/K206A/F217R/ 558N247D/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K 518 + + ++E40D/L44R/Y92H/K206A/F217R/ 559 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 519 + + ++ E40S/L44R/Y92H/K206A/F217R/ 560N247D/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K 520 + + +L44R/A77S/Y92H/K206A/F217R/ 561 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 521 + + ++ L44R/D52N/Y92H/K206A/F217R/ 562N247D/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K 522 + +++ ++L44R/E56K/Y92H/K206A/F217R/ 563 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 523 + + + L44R/N91M/Y92H/K206A/F217R/ 564N247D/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K 524 − + +L44R/N91V/Y92H/K206A/F217R/ 565 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 525 + ++ ++ L44R/Q76H/Y92H/K206A/F217R/ 566N247D/Q302K/L316D/M322I/ A337P/K362Q/E367N/W368A/R373K 526 + + ++L44R/R74H/Y92H/K206A/F217R/ 567 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 527 + + ++ L44R/Y92E/K206A/F217R/N247D/ 568Q302K/L316D/M322I/A337P/ K362Q/E367N/R373K 528 + + ++L44R/Y92H/D130Q/K206A/F217R/ 569 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 529 + + ++ L44R/Y92H/K182A/K206A/F217R/ 570N247D/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K 530 + + ++L44R/Y92H/K182E/K206A/F217R/ 571 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 531 + + ++ L44R/Y92H/K182H/K206A/F217R/ 572N247D/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K 532 + + ++L44R/Y92H/K182M/K206A/F217R/ 573 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 533 + + ++ L44R/Y92H/K182Q/K206A/F217R/ 574N247D/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K 534 + + ++L44R/Y92H/K182R/K206A/F217R/ 575 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 535 + + ++ L44R/Y92H/K182T/K206A/F217R/ 576N247D/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K 536 + + ++L44R/Y92H/K182V/K206A/F217R/ 577 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 537 + + ++ L44R/Y92H/K182Y/K206A/F217R/ 578N247D/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K 538 + + +L44R/Y92H/K206A/F217R/N247D/ 579 A287C/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 539 + + ++ L44R/Y92H/K206A/F217R/N247D/ 580A287H/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K 540 + + ++L44R/Y92H/K206A/F217R/N247D/ 581 A287M/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 541 + + ++ L44R/Y92H/K206A/F217R/N247D/ 582K283A/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K 542 + + ++L44R/Y92H/K206A/F217R/N247D/ 583 K283G/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 543 + + + L44R/Y92H/K206A/F217R/N247D/ 584K283M/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K 544 + + +L44R/Y92H/K206A/F217R/N247D/ 585 K283V/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 545 + + ++ L44R/Y92H/K206A/F217R/N247D/ 586K295A/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K 546 + +++ ++L44R/Y92H/K206A/F217R/N247D/ 587 K295E/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 547 + + ++ L44R/Y92H/K206A/F217R/N247D/ 588K295L/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K 548 + +++ ++L44R/Y92H/K206A/F217R/N247D/ 589 K295N/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 549 + ++ ++ L44R/Y92H/K206A/F217R/N247D/ 590K295Q/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K 550 + + ++L44R/Y92H/K206A/F217R/N247D/ 591 K295 S/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 551 + + ++ L44R/Y92H/K206A/F217R/N247D/ 592K295T/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K 552 + + ++L44R/Y92H/K206A/F217R/N247D/ 593 Q302K/L316D/A317D/M322I/A337P/K362Q/E367N/R373K 553 + + ++ L44R/Y92H/K206A/F217R/N247D/ 594Q302K/L316D/A317Q/M322I/ A337P/K362Q/E367N/R373K 554 + + ++L44R/Y92H/K206A/F217R/N247D/ 595 Q302K/L316D/M322I/A337P/A346G/K362Q/E367N/R373K 555 + + + L44R/Y92H/K206A/F217R/N247D/ 596Q302K/L316D/M322I/A337P/ G344A/K362Q/E367N/R373K 556 − − +L44R/Y92H/K206A/F217R/N247D/ 597 Q302K/L316D/M322I/A337P/G344D/K362Q/E367N/R373K 557 + + ++ L44R/Y92H/K206A/F217R/N247D/ 598Q302K/L316D/M322I/A337P/ G344S/K362Q/E367N/R373K 558 − − +L44R/Y92H/K206A/F217R/N247D/ 599 Q302K/L316D/M322I/A337P/I353L/K362Q/E367N/R373K 559 + + ++ L44R/Y92H/K206A/F217R/N247D/ 600Q302K/L316D/M322I/A337P/ K362Q/E367N/L372W/R373K 560 + ++ ++L44R/Y92H/K206A/F217R/N247D/ 601 Q302K/L316D/M322I/A337P/K362Q/E367N/W368A/R373K 561 + + ++ L44R/Y92H/K206A/F217R/N247D/ 602Q302K/L316D/M322I/A337P/ K362Q/E367N/W368L/R373K 562 + + ++L44R/Y92H/K206A/F217R/N247D/ 603 Q302K/L316D/M322I/A337P/K362Q/E367N/W368N/R373K 563 + + ++ L44R/Y92H/K206A/F217R/N247D/ 604Q302K/L316D/M322I/A337P/ K362Q/E367N/W368R/R373K 564 + + ++L44R/Y92H/K206A/F217R/N247D/ 605 Q302K/L316D/M322I/A337P/K362Q/E367N/W368V/R373K 565 + + ++ L44R/Y92H/K206A/F217R/N247D/ 606Q302K/L316D/M322I/A337P/ N348E/K362Q/E367N/R373K 566 + + ++L44R/Y92H/K206A/F217R/N247D/ 607 Q302K/L316D/M322I/A337P/N348M/K362Q/E367N/R373K 567 + + ++ L44R/Y92H/K206A/F217R/N247D/ 608Q302K/L316D/M322I/A337P/ N348Q/K362Q/E367N/R373K 568 + + ++L44R/Y92H/K206A/F217R/N247D/ 609 Q302K/L316D/M322I/A337P/N348R/K362Q/E367N/R373K 569 + + ++ L44R/Y92H/K206A/F217R/N247D/ 610Q302K/L316D/M322I/A337P/ N348W/K362Q/E367N/R373K 570 + + ++L44R/Y92H/K206A/F217R/N247D/ 611 Q302K/L316D/M322I/A337P/T354S/K362Q/E367N/R373K 571 + + ++ L44R/Y92H/K206A/F217R/N247D/ 612Q302K/N305K/L316D/M322I/ A337P/K362Q/E367N/R373K 572 + +++ ++L44R/Y92H/K206A/F217R/N247D/ 613 Q302K/N305L/L316D/M322I/A337P/K362Q/E367N/R373K 573 + + ++ L44R/Y92H/K206A/F217R/N247D/ 614Q302K/S314A/L316D/M322I/ A337P/K362Q/E367N/R373K 574 + + ++L44R/Y92H/K206A/F217R/N247D/ 615 Q302K/S314H/L316D/M322I/A337P/K362Q/E367N/R373K 575 + + ++ L44R/Y92H/K206A/F217R/N247D/ 616Q302K/S314N/L316D/M322I/ A337P/K362Q/E367N/R373K 576 + + ++L44R/Y92H/K206A/F217R/N247D/ 617 Q302K/S314Y/L316D/M322I/A337P/K362Q/E367N/R373K 577 + +++ ++ L44R/Y92H/K206A/F217R/W246A/ 618N247D/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K 578 + +++ ++L44R/Y92H/K206A/F217R/W246I/ 619 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 579 + +++ ++ L44R/Y92H/K206A/F217R/W246P/ 620N247D/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K 580 + +++ ++L44R/Y92H/K206A/F217R/W246R/ 621 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 581 + ++ ++ L44R/Y92H/K206A/F217R/W246S/ 622N247D/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K 582 + + ++L44R/Y92H/K206A/S210A/F217R/ 623 N247D/Q302K/L316D/M322I/A337P/A350T/K362Q/E367N/R373K 583 + + ++ L44R/Y92H/K206A/S210A/F217R/624 N247D/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K 584 + + ++L44R/Y92H/K206A/S210E/F217R/ 625 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 585 + + ++ L44R/Y92H/K206A/S210K/F217R/ 626N247D/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K 586 + ++ ++L44R/Y92H/K206A/S210N/F217R/ 627 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 587 + + ++ L44R/Y92H/K206A/S210R/F217R/ 628N247D/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K 588 + + +L44R/Y92H/K96A/K206A/F217R/ 629 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 589 + + + L44R/Y92H/K96W/K206A/F217R/ 630N247D/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K 590 + + +L44R/Y92H/P179M/K206A/F217R/ 631 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 591 + + ++ L44R/Y92H/R189K/K206A/F217R/ 632N247D/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K 592 + + ++L44R/Y92H/R189V/K206A/F217R/ 633 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 593 + + ++ L44R/Y92H/S95A/K206A/F217R/ 634N247D/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K 594 + + ++L44R/Y92H/S95E/K206A/F217R/ 635 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 595 + + + L44R/Y92H/T186A/K206A/F217R/ 636N247D/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K 596 + ++ ++L44R/Y92H/T186G/K206A/F217R/ 637 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 597 + − + L44R/Y92H/T186V/K206A/F217R/ 638N247D/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K 598 + + ++L44R/Y92H/Y120H/K206A/F217R/ 639 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 599 + + ++ L44R/Y92H/Y120S/K206A/F217R/ 640N247D/Q302K/L316D/M322I/ ++ A337P/K362Q/E367N/R373K 600 + + +L44R/Y92H/Y120S/K206A/F217R/ 641 N247D/Q302K/L316D/M322I/A337P/L341F/K362Q/E367N/R373K 601 − + + M39C/L44R/Y92H/K206A/F217R/ 642N247D/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K 602 + + ++M39E/L44R/Y92H/K206A/F217R/ 643 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 603 + + + M39R/L44R/Y92H/K206A/F217R/ 644N247D/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K 604 + + ++M39V/L44R/Y92H/K206A/F217R/ 645 N247D/Q302K/L316D/M322I/ ++A337P/K362Q/E367N/R373K 605 + + ++ T10P/L44R/Y92H/K206A/F217R/ 646N247D/Q302K/L316D/M322I/ ++ A337P/K362Q/E367N/R373K 606 + + ++ T10P;L44R; Y92H; R189L; K206A; 647 F217R; N247D; Q302K; L316D; M322I; A337P;K362Q; E367N; R373K 607 + + ++ T8L; L44R; Y92H; K206A; F217R; 648 N247D;Q302K; L316D; M322I; A337P; K362Q; E367N; R373K 608 + + + T8Q; L44R;Y92H; K206A; F217R; 649 N247D; Q302K; L316D; M322I; A337P; K362Q; E367N;R373K 1. Relative activity was calculated as activity of thevariant/activity of Rd3BB (SEQ ID NO: 13) (encoded by SEQ ID NO: 11). 2.− = <1.5 relative activity to Rd3BB (SEQ ID NO: 13); + = 1.5 to 5relative activity over Rd3BB (SEQ ID NO: 13); ++ = >5 to 10 relativeactivity over Rd3BB (SEQ ID NO: 13); and +++ = >10 relative activityover Rd3BB (SEQ ID NO: 13).

TABLE 2.8 Relative Activity of GLA Variants After No Challenge (NC) orChallenge at the Indicated pH or Condition SEQ Variant pH pH Amino aciddifferences relative to ID # NC 3.3 9.7 SEQ ID NO: 5 (WT GLA) NO: 609 +++ ++ L44R/S166P/K206A/F217R/N247D/ 650 Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 610 + + − L44R/S47T/Y92H/S166P/K206A/ 651F217R/N247D/M259E/Q302K/ L316D/M322I/A337P/K362Q/ E367N/R373K/M390Q611 + ++ ++ L44R/Y92H/S166P/K206A/F217R/ 652 N247D/Q302K/L316D/M322I/ +A337P/K362Q/E367N/R373K/M390Q 612 + ++ ++ L44R/Y92H/S166P/K206A/F217R/653 N247D/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K/M392T 613 + ++ ++L44R/S47N/Y92H/S166P/K206A/ 654 F217R/N247D/H271A/Q302K/L316D/M322I/A337P/K362Q/ E367N/R373K/M390Q 614 + ++ ++L44R/S47T/Y92H/S166P/K206A/ 655 F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 615 + ++ ++ L44R/S47N/Y92H/S166P/K206A/656 F217R/N247D/Q302K/L316D/ M322I/A337P/K362Q/E367N/ R373K/M390H616 + + ++ L44R/S47T/Y92H/S166P/K206A/ 657 F217R/N247D/M259W/H271A/Q302K/L316D/M322I/A337P/ K362Q/E367N/R373K/M390Q 617 + ++ ++L44R/Y92H/L136V/S166P/K206A/ 658 F217R/N247D/M259A/Q302K/L316D/M322I/A337P/K362Q/ E367N/R373K/M390Q 618 + ++ ++L44R/S47T/Y92H/S166P/K206A/ 659 F217R/N247D/H271A/Q302K/L316D/M322I/A337P/K362Q/ E367N/R373K 619 + ++ ++L44R/S47T/Y92H/S166P/K206A/ 660 F217R/N247D/H271A/Q302K/L316D/M322I/A337P/K362Q/ E367N/R373K/M390H 620 − + ++L44R/S47T/Y92H/S166P/K206A/ 661 F217R/N247D/H271A/Q302K/L316D/M322I/A337P/K362Q/E367N/ R373K/M390Q 621 + ++ −L44R/S47T/Y92H/S166P/K206A/ 662 F217R/N247D/M259E/H271A/Q302K/L316D/M322I/A337P/ K362Q/E367N/R373K/M390Q 622 + − ++L44R/S47N/Y92H/S166P/K206A/ F217R/N247D/M259W/H271A/Q302K/L316D/M322I/A337P/ 663 K362Q/E367N/R373K/M390Q/ M392T 623 + +++ ++L44R/S47N/S166P/K206A/F217R/ 664 N247D/H271A/A276S/Q302K/L316D/M322I/A337P/K362Q/E367N/ R373K/M392T 624 + ++ ++L44R/S47N/S166P/K206A/F217R/ 665 N247D/H271A/Q302K/L316D/M322I/A337P/K362Q/E367N/ R373K/M390Q 625 + ++ ++L44R/S47T/Y92H/S166P/K206A/ 44 F217R/N247D/H271A/Q302K/L316D/M322I/A337P/K362Q/E367N/ R373K/M392T 626 + ++ ++L44R/Y92H/S166P/K206A/F217R/ 666 N247D/H271A/Q302K/L316D/M322I/A337P/K362Q/E367N/ R373K/M390Q 627 + − ++L44R/S47N/Y92H/S166P/K206A/ 667 F217R/N247D/M259W/H271A/Q302K/L316D/M322I/A337P/ K362Q/E367N/R373K/M390H/ M392T 628 + + ++L44R/S47N/Y92H/S166P/K206A/ 668 F217R/N247D/H271A/Q302K/L316D/M322I/A337P/K362Q/ E367N/R373K/M390H 629 + ++ ++L44R/S47T/S166P/K206A/F217R/ 669 N247D/H271A/Q302K/L316D/M322I/A337P/K362Q/E367N/ R373K/M390Q 630 + − ++L44R/S47T/Y92H/S166P/K206A/ 670 F217R/N247D/M259W/H271A/Q302K/L316D/M322I/A337P/ K362Q/E367N/R373K/M390H 631 + + ++L44R/S47T/A53S/Y92H/S166P/ 671 K206A/F217R/N247D/H271A/Q302K/L316D/M322I/A337P/ K362Q/E367N/R373K/M390Q 632 + ++ ++L44R/S47N/Y92H/S166P/K206A/ 672 F217R/N247D/H271A/Q302K/L316D/M322I/A337P/K362Q/ E367N/R373K/M392T 633 + ++ ++E43D/L44R/Y92S/S166P/K206A/ 673 F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 634 + ++ ++ E43D/L44R/Y92E/S166P/K206A/674 F217R/N247D/Q302K/L316D/ M322I/A337P/K362Q/E367N/R373K 635 + ++ ++E43D/L44R/Y92H/S166P/K206A/ 675 F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 636 + + ++ E43D/L44R/Y92N/S166P/K206A/ 676F217R/N247D/Q302K/L316D/ M322I/A337P/K362Q/E367N/R373K 637 + + ++E43Q/L44R/Y92E/S166P/K206A/ 677 F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 1. Relative activity was calculated asactivity of the variant/activity of Rd3BB (SEQ ID NO: 13) (encoded bySEQ ID NO: 11). 2. Variant # 625 (Rd7BB) has the polynucleotide sequenceof SEQ ID NO: 43 and polypeptide sequence of SEQ ID NO: 44. 3. − = <1.5relative activity to Rd3BB (SEQ ID NO: 13); + = 1.5 to 5 relativeactivity over Rd3BB (SEQ ID NO: 13); ++ = >5 to 10 relative activityover Rd3BB (SEQ ID NO: 13); and +++ = >10 relative activity over Rd3BB(SEQ ID NO: 13).

TABLE 2.9 Relative Activity of GLA Variants After No Challenge (NC) orChallenge at the Indicated pH or Condition SEQ Variant pH pH Amino aciddifferences relative to ID # NC 3.5 7.5 SEQ ID NO: 5 (WT GLA) NO: 638 −− − T10P/L44R/S47T/Y92H/S166P/ 678 K206A/F217R/N247D/A261G/H271A/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K/ M392T 639 − − −M39E/L44R/S47T/Y92H/S166P/ 679 K206A/F217R/N247Y/H271A/Q302K/L316D/M322I/A337P/ K362Q/E367N/R373K/M392T 640 + + +T10P/M39E/E43D/L44R/S47T/ 680 Y92H/S166P/K206A/F217R/N247D/H271A/Q302K/L316D/ M322I/A337P/K362Q/E367N/ R373K/M392T 641 − − −T10P/M39E/L44R/S47T/Y92H/ 681 S166P/K206A/F217R/N247D/S266P/H271A/Q302K/L316D/ M322I/A337P/K362Q/E367N/ R373K/M392T 642 + + +T10P/E43D/L44R/S47T/Y92H/ 682 S166P/K206A/F217R/N247D/A261G/H271A/Q302K/N305L/ L316D/M322I/A337P/K362Q/E367N/W368A/R373K/M392T 643 + − + T10P/L44R/S47T/Y92H/S166P/ 683K206A/F217R/N247D/H271A/ Q302K/L316D/M322I/R325S/A337P/K362Q/E367N/R373K/ M392T 644 − − − L44R/S47T/Y92H/S166P/K206A/ 684F217R/L237P/N247D/H271A/ Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392T 645 + + − L44R/S47T/Y92H/S166P/P174S/ 685K206A/F217R/N247D/H271A/ Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392T 646 − − − L44R/S47T/Y92H/G113C/S166P/ 686K206A/F217R/N247D/H271A/ Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392T 647 − − − L14F/L44R/S47T/Y92H/S166P/ 687K206A/F217R/N247D/H271A/ Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392T 648 + + + T10P/M39E/L44R/S47T/Y92H/ 46S166P/K206A/F217R/N247D/ A261G/H271A/Q302K/L316D/M322I/A337P/K362Q/E367N/ W368A/R373K/M392T 649 + + +T10P/M39E/L44R/S47T/Y92H/ 689 S166P/K206A/F217R/W246P/N247D/H271A/Q302K/L316D/ M322I/A337P/K362Q/E367N/ R373K/M392T 650 + + +R7H/T10P/L44R/S47T/Y92H/ 690 S166P/K206A/F217R/N247D/H271A/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K/ M392T 651 + + +T10P/L44R/S47T/Y92H/S166P/ 691 K206A/F217R/N247D/H271A/Q302K/L316D/M322I/A337P/ K362Q/E367N/R373K/M392T 652 + + −L44R/S47T/Y92H/S166P/K206A/ 692 F217R/W246P/N247D/A261G/H271A/Q302K/N305L/L316D/ M322I/A337P/K362Q/E367N/ R373K/M392T 653 + + +T10P/L44R/S47T/Y92H/S166P/ 693 K206A/F217R/W246P/N247D/H271A/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K/ M392T 654 + + + R7S/L44R/S47T/Y92H/S166P/ 694 K206A/F217R/N247D/H271A/Q302K/L316D/M322I/A337P/ K362Q/E367N/R373K/M392T 655 + − −L44R/S47T/Y92H/S166P/K206A/ 695 F217R/W246P/N247D/A261G/H271A/Q302K/N305L/L316D/ M322I/A337P/K362Q/E367N/ W368A/R373K/M392T656 + + + T10P/L44R/S47T/Y92H/S166P/ 696 K206A/F217R/W246P/N247D/A261G/H271A/Q302K/L316D/ M322I/A337P/K362Q/E367N/ R373K/M392T 657 + + +L44R/S47T/P67T/Y92H/S166P/ 697 K182N/K206A/F217R/N247D/H271A/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K/ M392T 658 + + +M39E/L44R/S47T/Y92H/S166P/ 698 K206A/F217R/W246P/N247D/H271A/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K/ M392T 659 − − −L44R/S47T/W64L/Y92H/S166P/ 699 K206A/F217R/N247D/H271A/Q302K/L316D/M322I/A337P/ K362Q/E367N/R373K/M392T 660 + + −M39E/L44R/S47T/Y92H/S166P/ 700 K206A/F217R/N247D/A261G/H271A/Q302K/N305L/L316D/ M322I/A337P/K362Q/E367N/ R373K/M392T 661 − − −L44R/S47T/Y92H/S166P/W195C/ 701 K206A/F217R/N247D/H271A/Q302K/L316D/M322I/A337P/ K362Q/E367N/R373K/M392T 662 + + +L44R/S47T/Y92H/S166P/K206A/ 702 F217R/V238I/N247D/H271A/Q302K/L316D/M322I/A337P/ K362Q/E367N/R373K/M392T 663 + + −E43D/L44R/S47T/Y92H/S166P/ 703 K206A/F217R/N247D/A261G/H271A/Q302K/L316D/M322I/ A337P/K362Q/E367N/W368A/ R373K/M392T 664 − − −T10P/L44R/S47T/Y92H/S166P/ 704 K206A/F217R/N247D/Q252H/M253R/A254E/A261G/H271A/ Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392T 665 + + + R7C/L44R/S47T/Y92H/S166P/ 705K206A/F217R/N247D/H271A/ Q302K/L316D/M322I/A337P/K362Q/E367N/W368A/R373K/ M392T 666 + + − L44R/S47T/Y92H/S166P/K206A/ 706F217R/P228L/N247D/H271A/ Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392T 667 + + + D30G/L44R/S47T/Y92H/S166P/ 707K206A/F217R/N247D/H271A/ Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392T 668 + + + M39E/L44R/S47T/Y92H/S166P/ 708K206A/F217R/N247D/H271A/ Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392T 669 + − − L44R/S47T/Y92H/S166P/K206A/ 709F217R/N247D/P262S/H271A/ Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392T 670 + + + L44R/S47T/Y92H/S166P/K206A/ 710F217R/N247D/H271A/Q302K/ N305L/L316D/M322I/A337P/K362Q/E367N/R373K/M392T 671 ++ ++ + T10P/L44R/S47T/Y92H/S166P/ 711K206A/F217R/N247D/H271A/ Q302K/L316D/M322I/A337P/K362Q/E367N/W368A/R373K/ M392T 672 + + − L44R/S47T/Y92H/D144Y/S166P/ 712K206A/F217R/N247D/H271A/ Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392T 673 + + − L44R/S47T/Y92H/S166P/K206A/ 713F217R/N247D/H271A/Q302K/ L316D/M322I/A337P/K362Q/E367N/R373K/N377Y/M392T 674 − − − L44R/S47T/Y92H/S166P/K206A/ 714F217R/P234H/N247D/H271A/ Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392T 675 + + − L44R/S47T/M65V/Y92H/S166P/ 715K206A/F217R/N247D/H271A/ Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392T 676 + + + M39E/L44R/S47T/Y92H/S166P/ 716K206A/F217R/N247D/H271A/ Q302K/L316D/M322I/A337P/K362Q/E367N/W368A/R373K/ M392T 677 + + − L44R/S47T/Y92H/S166P/K206A/ 717F217R/N247D/M253W/H271A/ S273D/P274S/K277R/Q302K/L316D/M322I/A337P/K362Q/ E367N/R373K/M392T 678 − − −L44R/S47T/Y92H/S166P/K206A/ 718 F217R/N247D/M253W/A257G/H271A/K277R/Q281L/Q302K/ L316D/A319D/M322I/A337P/K362Q/E367N/R373K/M392T 679 + + + T10P/M39E/L44R/S47T/Y92H/ 719S166P/K206A/F217R/N247D/ H271A/Q302K/N305L/L316D/M322I/A337P/K362Q/E367N/ R373K/M392T 680 + + + T10P/M39E/L44R/S47T/Y92H/720 S166P/K206A/F217R/N247D/ H271A/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/ M392T 681 − − − R7P/L44R/S47T/Y92H/S166P/ 721K206A/F217R/N247D/H271A/ Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392T 682 + + + L44R/S47T/Y92H/S166P/K206A/ 722F217R/N247D/H271A/ Q302K/L316Y/M322I/A337P/ K362Q/E367N/R373K/M392T683 + + − M39E/E43D/L44R/S47T/Y92H/ 723 S166P/K206A/F217R/W246P/N247D/M253W/H271A/S273D/ Q302K/L316D/M322I/A337P/K362Q/E367N/W368A/R373K/M392T 684 + + + L44R/S47T/Y92H/S166P/K206A/ 724F217R/N247D/H271A/Q302K/ N305L/L316D/M322I/A337P/K362Q/E367N/W368A/R373K/ M392T 685 + − − E43D/L44R/S47T/Y92H/S166P/ 725K206A/F217R/N247D/M253W/ A257G/H271A/Q302K/N305L/L316D/M322I/A337P/K362Q/ E367N/W368A/R373K/M392T 686 − − +T10P/E17G/L44R/S47T/Y92H/ 726 S166P/K206A/F217R/N247D/H271A/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K/ M392T 687 + − −L44R/S47T/Y92H/S166P/K206A/ 727 F217R/N247D/H271A/Q290R/Q302K/L316D/M322I/A337P/ K362Q/E367N/W368A/R373K/ M392T 688 + + −L44R/S47T/Y92H/S166P/K206A/ 728 F217R/P228Q/N247D/H271A/Q302K/L316D/M322I/A337P/ K362Q/E367N/R373K/M392T 689 + + +L44R/S47T/Y92H/S166P/K206A/ 729 F217R/N247D/H271A/Q302K/N305L/L316D/M322I/A337P/ K362Q/E367N/R373K/M392T 690 + − −T10P/L44R/S47T/Y92H/M156V/ 730 S166P/K206A/F217R/N247D/H271A/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K/ M392T 691 + + +T10P/L44R/S47T/Y92H/S166P/ 731 K206A/F217R/N247D/H271A/Q302K/L316D/M322I/A337P/ K362Q/E367N/R373K/M392T 692 − − −L44R/S47T/Y92H/S166P/K206A/ 732 F217R/N247D/W256L/H271A/Q302K/L316D/M322I/A337P/ K362Q/E367N/R373K/M392T 693 + + +L44R/S47T/Y92H/S166P/K206A/ 733 F217R/N247D/H271A/Q302K/L316D/M322I/A337P/K362Q/ E367N/W368A/R373K/M392T 1. Relative activitywas calculated as activity of the variant/activity of Rd7BB (SEQ ID NO:44) (encoded by SEQ ID NO: 43). 2. − = <0.5 relative activity to Rd7BB(SEQ ID NO: 44); + = >0.5 to 1.5 relative activity over Rd7BB (SEQ IDNO: 44); and ++ = >1.5 relative activity over Rd7BB (SEQ ID NO: 44);

Example 3 In Vitro Characterization of GLA Variants Production of GLA inYeast

In order to produce GLA-containing supernatant, replica HTP-cultures ofGLA were grown as described in Example 2. Supernatants from replicacultures (n=12-36) were combined prior to further analysis.

Production of GLA in HEK293T Cells

Secreted expression of GLA variants in mammalian cells was performed bytransient transfection of HEK293 cells. Cells were transfected with GLAvariants (SEQ ID NOS:3, 4, 9, 12, 17, 20, 23, and 41) fused to anN-terminal synthetic mammalian signal peptide and subcloned into themammalian expression vector pLEV113 as described in Example 1. HEK293cells were transfected with plasmid DNA and grown in suspension for 4days using techniques known to those skilled in the art. Supernatantswere collected and stored at 4° C.

Example 4 Purification of GLA Variants Purification of GLA Variants FromMammalian Cell Supernatants

GLA variants were purified from mammalian culture supernatantessentially as known in the art (See, Yasuda et al., Prot. Exp. Pur.,37, 499-506 [2004]). Concanavalin A resin (Sigma Aldrich) wasequilibrated with 0.1 M sodium acetate, 0.1 M NaCl, 1 mM MgCl₂, CaCl₂,and MnCl₂ pH 6.0 (Con A binding buffer). Supernatant was diluted 1:1with binding buffer and loaded onto the column. The column was washedwith 15 volumes of Concanavalin A binding buffer, and samples wereeluted by the addition of Concanavalin A binding buffer including 0.9 Mmethyl-α-D-mannopyranoside and 0.9 M methyl-α-D-glucopyranoside. Elutedprotein was concentrated and buffer exchanged three times using aCentricon® Plus-20 filtration unit with a 10 kDa molecular weight cutoff (Millipore) into ThioGal binding buffer (25 mM citrate-phosphate,0.1 M NaCl, pH 4.8). Buffer exchanged samples were loaded onto aImmobilized-D-galactose resin (Pierce) equilibrated with ThioGal bindingbuffer. The resin was washed with six volumes of ThioGal binding bufferand eluted with 25 mM citrate phosphate, 0.1 M NaCl, 0.1 M D-galactose,pH 5.5. Eluted samples were concentrated using a Centricon® Plus-20filtration unit with a 10 kDa molecular weight cut off. Purificationresulted in between 2.4-10 μg of purified protein/ml of culturesupernatant based on Bradford quantitation.

SDS-PAGE Analysis of GLA Variants

Samples of yeast culture supernatant and mammalian cell culturesupernatant and purified GLA were analyzed by SDS-PAGE. In the yeastsupernatants, GLA levels were too low to be detected via this method.Bands corresponding to the ˜49 kDa predicted GLA molecular weight werefound in both mammalian cell culture supernatants and purified GLAsamples.

Immunoblot Analysis of GLA Variants

Samples of yeast supernatant and mammalian cell culture supernatant wereanalyzed by immunoblot. Briefly, samples were separated via SDS-PAGE.Protein was transferred to a PVDF membrane using an iBlot dry blotsystem (Life Technologies). The membrane was blocked with Odysseyblocking buffer (TBS) (LI-COR) for 1 h at RT and probed with a 1:250dilution of rabbit a-GLA IgG (Thermo-Fischer) in Odyssey blocking bufferwith 0.2% Tween® 20 for 14 h at 4° C. The membrane was washed 4×5 minwith Tris-buffered saline+0.1% Tween® 20 and probed with a 1:5000dilution of IRDye800CW donkey a-rabbit IgG (LI-COR) in Odyssey blockingbuffer with 0.2% Tween® 20 and 0.01% SDS for 1 hr at RT. The membranewas washed 4×5 mM with Tris-buffered saline+0.1% Tween® 20, and analyzedusing an Odyssey Imager (LI-COR). Bands corresponding to the ˜49 kDapredicted GLA molecular weight were found in both the mammalian cellculture and yeast supernatants. In S. cerevisiae expressed samples,mutants containing the mutation E367N ran at a slightly higher MW. Thismutation introduces a canonical NXT N-linked glycosylation site (where Xis any amino acid except P) and the possible introduction of anadditional N-linked glycan may account for the higher MW.

Example 5 In Vitro Characterization of GLA Variants

Optimization of Signal Peptide for Secreted Expression of GLA by S.cerevisiae

S. cerevisiae transformed with Mfleader-GLA (SEQ ID NO:7), SP-GLA (SEQID NO:36) or a vector control were grown in HTP as described in Example2. Cultures were grown for 48-120 h prior to harvest of the supernatantand analysis (n=6) as described in Example 2. FIG. 1 provides a graphshowing the relative activity of different GLA constructs in S.cerevisiae after 2-5 days of culturing. As indicated in this Figure,SP-GLA (SEQ ID NO:36) produced a high level of active enzyme thatsaturated after three days of growth.

pH Stability of GLA Variants Expressed in S. cerevisiae

GLA variants were challenged with different buffers to assess theoverall stability of the enzyme. First, 50 μL of supernatant from a GLAvariant yeast culture and 50 uL of McIlvaine buffer (pH 2.86-9.27) or200 mM sodium carbonate (pH 9.69) were added to the wells of a 96-wellround bottom plate (Costar #3798, Corning). The plates were sealed andincubated at 37° C. for 1 h. For the assay, 50 μL of challengedsupernatant was mixed with 25 μL of 1 M citrate buffer pH 4.3 and 25 μLof 4 mM MUGal in McIlvaine buffer pH 4.8. The reactions were mixedbriefly and incubated at 37° C. for 60-180 minutes, prior to quenchingwith 100 μL of 1 M sodium carbonate. Hydrolysis was analyzed using aSpectraMax® M2 microplate reader monitoring fluorescence (Ex. 355 nm,Em. 448 nm). FIG. 2 provides graphs showing the absolute (Panel A) andrelative (Panel B) activity of GLA variants after incubation at variouspHs.

Thermostability of GLA Variants Expressed in S. cerevisiae

GLA variants were challenged at various temperatures in the presence andabsence of 1 μM 1-deoxygalactonojirimycin (Migalastat; Toronto ResearchChemicals) to assess the overall stability of the enzyme. First, 50 μLof supernatant from a GLA variant yeast culture and 50 uL of McIlvainebuffer (pH 7.65)+/−2 mM 1-deoxygalactonojirimycin were added to thewells of a 96-well PCR plate (Biorad, HSP-9601). The plates were sealedand incubated at 30-54° C. for 1 h using the gradient program of athermocycler. For the assay, 50 μL of challenged supernatant was mixedwith 25 μL of 1 M citrate buffer pH 4.3 and 25 μL of 4 mM MUGal inMcIlvaine buffer pH 4.8. The reactions were mixed briefly and incubatedat 37° C. for 90 minutes, prior to quenching with 100 μL of 1 M sodiumcarbonate. Hydrolysis was analyzed using a SpectraMax® M2 microplatereader monitoring fluorescence (Ex. 355 nm, Em. 448 nm). FIG. 3 providesgraphs showing the absolute (Panel A) and relative (Panel B) activity ofGLA variants after incubation at various temperatures.

Serum Stability of GLA Variants Expressed in S. cerevisiae

To assess the relative stability of variants in the presence of blood,samples were exposed to serum. First, 20 μL of supernatant from a GLAvariant yeast culture and 0-80 μL of water and 0-80 μL of bovine serumwere added to the wells of a 96-well round bottom plate (Costar #3798,Corning). The plates were sealed and incubated at 37° C. for 1 h. Forthe assay, 50 μL of challenged supernatant was mixed with 25 μL of 1 Mcitrate buffer pH 4.3 and 25 μL of 4 mM MUGal in McIlvaine buffer pH4.8. The reactions were mixed briefly and incubated at 37° C. for 90minutes, prior to quenching with 100 μL, of 1 M sodium carbonate.Hydrolysis was analyzed using a SpectraMax® M2 microplate readermonitoring fluorescence (Ex. 355 nm, Em. 448 nm). FIG. 4 provides graphsshowing the absolute (Panels A and B) and relative (Panels C and D)activity of GLA variants after challenge with various percentages ofserum.

Relative Activities of GLA Variants Expressed in HEK293T Cells

Supernatants from GLA variants expressed in HEKT293T cells were seriallydiluted 2× with supernatant from an non GLA expressing yeast culture.Dilutions (50 μL) were mixed with 25 μL of 4 mM MUGal in McIlvaineBuffer pH 4.8 and 25 μL of 1 M citrate buffer pH 4.3 in a Corning®96-well, black, opaque bottom plate. The reactions were mixed brieflyand incubated at 37° C. for 60 minutes, prior to quenching with 100 μLof 1 M sodium carbonate. Hydrolysis was analyzed using a SpectraMax® M2microplate reader monitoring fluorescence (Ex. 355 nm, Em. 448 nm). FIG.5 provides a graph showing the relative activity of GLA variantsexpressed in HEK293T cells. Supernatants from cells transfected withvariant GLA enzymes showed markedly higher hydrolase activities comparedto the WT enzymes, and much more activity per volume than was seen in S.cerevisiae expression.

pH Stability of GLA Variants Expressed in HEK293T Cells

GLA variants were challenged with different buffers to assess theiroverall stability. Supernatants from mammalian cell cultures werenormalized to equal activities by dilution with supernatant from a nonGLA expressing culture. Normalized supernatants (50 μL) and 50 uL ofMcIlvaine buffer (pH 4.06-8.14) were added to the wells of a 96-wellround bottom plate (Costar #3798, Corning). The plates were sealed andincubated at 37° C. for 3 h. For the assay, 50 μL of challengedsupernatant was mixed with 25 μL of 1 M citrate buffer pH 4.3 and 25 μLof 4 mM MUGal in McIlvaine buffer pH 4.8. The reactions were mixedbriefly and incubated at 37° C. for 3 h, prior to quenching with 100 μLof 1 M sodium carbonate. Hydrolysis was analyzed using a SpectraMax® M2microplate reader monitoring fluorescence (Ex. 355 nm, Em. 448 nm). FIG.6 provides graphs showing the absolute (Panel A) and relative (Panel B)activity of GLA variants expressed in HEK293T cells, normalized foractivity, and incubated at various pHs.

All enzymes were found to be more stable versus pH challenges whencompared to WT GLA expressed in S. cerevisiae (compare with FIG. 2).This difference is possibly due to differential glycosylation betweenexpression hosts. However, it is not intended that the present inventionbe limited to any particular mechanism or theory. Mutant enzymes hadbroader pH stability profiles compared to the WT enzyme expressed inHEK293T.

Thermostability of GLA Variants Expressed in HEK293T Cells

GLA variants were challenged at various temperatures in the presence andabsence of 1 μM 1-deoxygalactonojirimycin (Migalastat) to assess theiroverall stability. Supernatants from mammalian cell cultures werenormalized to approximately equal activities by dilution withsupernatant from a non GLA expressing culture. Diluted supernatants wereadded to the wells of a 96-well PCR plate (Biorad, HSP-9601). The plateswere sealed and incubated at 30-54° C. for 1 h using the gradientprogram of a thermocycler. For the assay, 20 μL of challengedsupernatant was mixed with 30 μL of 1 M citrate buffer pH 4.3 and 50 μLof 4 mM MUGal in McIlvaine buffer pH 4.8. The reactions were mixedbriefly and incubated at 37° C. for 90 minutes, prior to quenching with100 μL of 1 M sodium carbonate. Hydrolysis was analyzed using aSpectraMax® M2 microplate reader monitoring fluorescence (Ex. 355 nm,Em. 448 nm). FIG. 7 provides graphs showing the absolute (Panel A) andrelative (Panel B) activity of GLA variants expressed in HEK293T cells,normalized for activity, and incubated at various temperatures. As shownin this Figure, all of the enzymes were more stable after temperaturechallenges when compared to WT GLA expressed in S. cerevisiae (comparewith FIG. 2), likely due to differential glycosylation betweenexpression hosts. In the GLA variants (SEQ ID NOS:10 and 13) the T_(m)of the enzyme was increased by 2 and 4° C. respectively. Addition ofMigalastat increased the T_(m) by 5.5° C., however at a 0.2 μM finalconcentration in the assay, activity in the Migalastat treated samplewas reduced by ˜60%.

Activity of WT GLA and GLA Variants on an Alternative Substrate

To confirm that improved activity in MUGal hydrolysis corresponded tomore native substrates, mammalian cell-expressed GLA variants wereassayed using N-Dodecanoyl-NBD-ceramide trihexoside (NBD-GB3) assubstrate. HEK293T culture supernatant (10 μL), 100 mM sodium citrate pH4.8 (80 μL), and NBD-GB3 (0.1 mg/ml) in 10% ethanol (10 μL) were addedto microcentrifuge tubes. Samples were inverted to mix, and incubated at37° C. for 1 h. The reaction was quenched via addition of 50 μLmethanol, diluted with 100 μL chloroform, vortexed and the organic layerwas isolated for analysis. The organic phase (10 μL) was spotted onto asilica plate and analyzed by thin layer chromatography(chloroform:methanol:water, 100:42:6), detecting the starting materialand product using a 365 nm UV lamp. Significant conversion was observedonly with SEQ ID NO:13, confirming that the variant exhibits improvedactivity, as compared to the WT GLA.

Specific Activity of GLA Variants

GLA variants purified as described in Example 4, were evaluated fortheir specific activity. Between 0-0.25 ng of purified enzyme was addedto 4 mM MUGal in McIlvaine buffer pH 4.8 (final pH of 4.8). Samples wereincubated for 60 min at 37° C. and quenched via addition of 100 μL, of 1M sodium carbonate. Hydrolysis was analyzed using a SpectraMax® M2microplate reader monitoring fluorescence (Ex. 355 nm, Em. 448 nm), andcorrelated to absolute amounts of 4-methylumbelliferone through the useof a standard curve.

pH Stability of Purified GLA Variants Over Time

To confirm that purified GLA variants show the desired pH stabilityobserved after expression in yeast, WT GLA (SEQ ID NO:5) and SEQ IDNO:42 were incubated in acidic or basic buffers and analyzed forresidual activity. GLA variants (200 ng) were added to McIlvaine bufferpH 4.1 or 7.5 and incubated for 0-24 h at 37° C. Samples (50 μL) wereadded to a mixture of 25 uL 1M citric acid pH 4.3 and 25 μL of 4 mMMUGal in McIlvaine buffer pH 4.8, and incubated at 37° C. for 1 h.Samples were quenched with 100 μL of 1 M sodium carbonate, diluted 1:4in 1 M sodium carbonate and analyzed by fluorescence spectroscopy (Ex.355, Em. 448). SEQ ID NO:42 was considerably more stable under bothacidc and basic challenge conditions confirming that stability advancesdeveloped in yeast translated to the protein expressed in mammaliancells (See FIG. 8 for graphs of the results).

Thermostability of Purified GLA Variants Expressed in HEK293T Cells

The thermostability of WT GLA (SEQ ID NO:5) and SEQ ID NO:42 weredetermined to assess their overall stability. Purified enzyme asdescribed in Example 4 was diluted to 20 μg/ml in 1×PBS with 1×SyproOrange (Thermo Fischer Scientific), and added to a 96-well PCR plate(Biorad, HSP-9601). The plates were heated from 30 to 75° C. at 0.5°C./min on a RT-PCR machine and Sypro Orange fluorescence was monitored.Under these conditions WT GLA melted at 37° C., while SEQ ID NO:42melted at 55° C.

Example 6 In Vivo Characterization of GLA Variants SerumPharmacokinetics of Purified GLA Variants

Purified GLA variants produced as described in Example 4 were assessedfor stability in the serum of live rats. WT GLA (SEQ ID NO:5) or SEQ IDNO:42 at 1 mg/ml were administered intravenously at 1 ml/kg to threenaïve jugular vein cannulated Sprague-Dawley rats (7-8 weeks old) each.Prior to administration and at 5, 15, 30, 60, 120, and 240 minutespost-administration, 200 μL of blood was collected from each rat in anEDTA tube and centrifuged at 4° C. and 6000 rpm to generate >80 μL ofserum per sample. Samples were frozen and stored on dry ice prior toanalysis. For analysis, serum (10 μL) was added to 40 μL of 5 mM MUGalin McIlvaine buffer pH 4.4, and incubated at 37° C. for 1 h. Sampleswere quenched with 50 μL of 1 M sodium carbonate, diluted 1:100 in 1 Msodium carbonate and analyzed by fluorescence spectroscopy (Ex. 355, Em.448). Four hours post-administration SEQ ID NO:42 retained 15.3% ofmaximal activity, while WT GLA retained only 0.66% (See, FIG. 9).

Example 7 Deimmunization of GLA

In this Example, experiments conducted to identify diversity that wouldremove predicted T-cell epitopes from GLA are described.

Identification of Deimmunizing Diversity:

To identify mutational diversity that would remove T-cell epitopes,computational methods were used to identify GLA subsequences that werepredicted to bind efficiently to representative HLA receptors. Inaddition, experimental searches for amino acid mutations were conducted,particularly for mutations that do not affect GLA activity (e.g., in theassays described in Example 2). The amino acid sequences of activevariants were then analyzed for predicted immunogenicity usingcomputational methods.

Computational Identification of Putative T-Cell Epitopes in a WT GLA:

Putative T-cell epitopes in a WT GLA (SEQ ID NO:5) were identified usingthe Immune Epitope Database (IEDB; Immune Epitope Database and AnalysisResource website) tools, as known in the art and proprietary statisticalanalysis tools (See e.g., iedb.org and Vita et al., Nucl. Acids Res.,38(Database issue):D854-62 [2010]. Epub 2009 Nov. 11]). The WT GLA wasparsed into all possible 15-mer analysis frames, with each frameoverlapping the last by 14 amino acids. The 15-mer analysis frames wereevaluated for immunogenic potential by scoring their 9-mer core regionsfor predicted binding to eight common class II HLA-DR alleles(DRB1*0101, DRB1*0301, DRB1*0401, DRB1*0701, DRB1*0801, DRB1*1101,DRB1*1301, and DRB1*1501) that collectively cover nearly 95% of thehuman population (See e.g., Southwood et al., J. Immunol., 160:3363-3373[1998]), using methods recommended on the IEDB website. Potential T-cellepitope clusters contained within the enzyme (i.e., sub-regionscontained within GLA which have an unusually high potential forimmunogenicity) were identified using statistical analysis tools, asknown in the art. The identified T-cell epitope clusters were screenedagainst the IEDB database of known epitopes. These screens identifiedfive putative T-cell epitopes in the WT enzyme. These epitopes arereferred to as TCE-I, II, III, IV, and V below.

Design of Deimmunizing Libraries:

First, the sequences of active GLA mutants identified in Example 2 areassessed for the presence of T-cell epitopes. Mutations identified topotentially reduce binding to the HLA-DR alleles are incorporated into arecombination library. Additional libraries are prepared usingsaturation mutagenesis of every single amino acid within the five T-cellepitopes. Hits from these libraries are subjected to further rounds ofsaturation mutagenesis, HTP screening, and recombination to remove allpossible T-cell epitopes.

Construction and Screening of Deimmunizing Libraries:

Combinatorial and saturation mutagenesis libraries designed as describedabove were constructed by methods known in the art, and tested foractivity in an unchallenged assay as described in Example 2. Activevariants were identified and sequenced. Their activities and mutationswith respect to WT GLA are provided in the table below.

Identification of Deimmunizing Diversity:

Active variants were analyzed for their levels of predictedimmunogenicity by evaluating their binding to the eight common Class IIHLA-DR alleles as described above. The total immunogenicity score andimmunogenic hit count are shown in Table 7.1. The total immunogenicityscore (TIS) reflects the overall predicted immunogenicity of the variant(i.e., a higher score indicates a higher level of predictedimmunogenicity). The immunogenic “hit count” (IHC) indicates the numberof 15-mer analysis frames with an unusually high potential forimmunogenicity (i.e., a higher score indicates a higher potential forimmunogenicity). Mutations resulting in a lower total immunogenicityscore and/or an immunogenic hit count less than that of the referencesequence were considered to be potential “deimmunizing mutations”. Acollection of the most deimmunizing mutations were recombined togenerate a number of variants that were active and predicted to besignificantly less immunogenic than WT GLA. In the following Table,total immunogenicity score (TIS) and immunogenic hit count (IHC) areprovided.

TABLE 7.1 Immunogenicity Score (TIS), and Immunogenic Hit Count (IHC)for GLA Variants Var- SEQ iant ID # NO: Active Mutations TIS IHC 5 WTGLA 450 38 33 79 A199H/E367S 468 47 34 80 A337P 444 38 1 47 A337S 449 3835 81 A339S 450 38 36 82 A350G 450 38 296 337A66T/K206A/F217R/L316D/M322I/ 429 38 A337P/K343G/A350G/E367N/R373K 200244 C143A/K206A 429 38 201 245 C143T/K206A 429 38 202 246 C59A/K206A 42738 37 83 D105A 458 38 38 84 D105S 462 38 39 85 D124N/E147G/N161K/R162Q/425 35 T163V/R165A/I167S/V168I/ Y169V/S170-/M177S/F217E 516 557D2E/L44R/Y92H/K206A/F217R/ 386 24 N247D/Q302K/L316D/M322I/Q326G/A337P/K362Q/E367N/R373K 517 558 D2Q/L44R/Y92H/K206A/F217R/ 393 24N247D/Q302K/L316D/ M322I/A337P/K362Q/E367N/R373K 667 707D30G/L44R/S47T/Y92H/S166P/ 345 8 K206A/F217R/N247D/H271A/Q302K/L316D/M322I/A337P/K362Q/ E367N/R373K/M392T 40 86 D396R 451 38 4187 D396T 452 38 42 88 E367N 462 43 43 89 E367T 462 45 44 90 E387K 460 3845 91 E387Q 457 38 46 92 E387R 457 38 47 93 E387T 459 38 48 94 E40D 44533 518 560 E40D/L44R/Y92H/K206A/F217R/ 390 24 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 519 561 E40S/L44R/Y92H/K206A/F217R/ 407 25N247D/Q302K/L316D/ M322I/A337P/K362Q/E367N/R373K 2 48 E43D 450 37 3 49E43D/E48D 449 37 4 50 E43D/E48D/I208V/N247D/Q299R/ 434 36Q302K/R373K/I376V 5 51 E43D/E48D/I208V/R373K 429 36 6 52E43D/E48D/I208V/R373K/I376V 428 36 7 53 E43D/E48D/N247D/Q299R/Q302K/ 44836 R373K/I376V 8 54 E43D/E48D/N247D/Q302K/R373K 442 36 9 55E43D/E48D/Q302K/R373K/I376V 442 36 10 56 E43D/I208V/N247D 435 37 11 57E43D/I208V/N247D/Q299R/R373K/I376V 435 36 12 58E43D/I208V/Q299R/R373K/I376V 436 36 663 703E43D/L44R/S47T/Y92H/S166P/K206A/ 315 1 F217R/N247D/A261G/H271A/Q302K/L316D/M322I/A337P/K362Q/E367N/W368A/ R373K/M392T 685 725E43D/L44R/S47T/Y92H/S166P/K206A/ 334 7 F217R/N247D/M253W/A257G/H271A/Q302K/N305L/L316D/M322I/A337P/K362Q/ E367N/W368A/R373K/M392T 634 674E43D/L44R/Y92E/S166P/K206A/ 362 21 F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 635 375 E43D/L44R/Y92H/S166P/ 378 21K206A/F217R/N247D/Q302K/ L316D/M322I/A337P/K362Q/E367N/R373K 636 376E43D/L44R/Y92N/S166P/K206A/ 366 21 F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 633 673 E43D/L44R/Y92S/S166P/K206A/365 21 F217R/N247D/Q302K/ L316D/M322I/A337P/K362Q/E367N/R373K 13 59E43D/N247D/R373K/I376V 442 36 14 60 E43D/R373K/I376V 443 36 637 377E43Q/L44R/Y92E/S166P/K206A/ 370 21 F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 15 61 E48D/I208V/Q299R/Q302K/R373K437 37 16 62 E48D/R373K/I376V 443 37 17 63 E48G/R373K 444 37 49 95 F180R454 38 50 96 F180S 449 38 51 97 F198S 450 38 52 98 F217D 450 38 53 99F217R 450 38 18 64 F217S 452 38 54 100 F352I 450 38 55 101 F352V/F365I447 38 56 102 F365I 447 38 57 103 F365K 446 38 58 104 F365L 448 38 59105 F365R 447 38 60 106 F365T 436 38 61 107 F365V 447 38 62 108G303Q/R373V 465 38 63 109 H155A 451 38 64 110 H155L 455 41 65 111 H155R452 39 66 112 H155T 449 38 213 255 H15Q/K206A 429 38 67 113 H375E 437 3668 114 H84S 450 38 69 115 I102L 450 38 70 116 I102L/L394V 449 37 71 117I123T/T369N 449 38 72 118 I167V 438 37 19 651208V/N247D/Q299R/Q302K/R373K/I376V 435 37 20 66I208V/N247D/Q299R/R373K/I376V 435 37 21 67 I208V/N247D/R373K/I376V 42837 22 68 I208V/Q299R/I376V 436 37 23 69 I208V/Q302K/R373K/I376V 429 3724 70 I376V 443 37 73 10 K206A 429 38 196 240 K206A/A350G 429 38 197 241K206A/A350G/K362Q/T369A 413 38 198 242 K206A/A350G/T369D 426 38 199 243K206A/A350G/T369S 429 38 203 247 K206A/E367A/T369D 439 42 204 248K206A/E367D 427 38 205 21 K206A/E367D/T369D 419 37 206 18 K206A/E367N441 43 207 249 K206A/E367N/R373K 430 38 208 250 K206A/E367N/R373K/I376V429 38 209 251 K206A/E367P/T369D 430 38 297 338K206A/F217R/G230V/N247D/Q302K/ 453 42 M322I/E367N/T369S/R373K 233 274K206A/F217R/N247D/L316D/ 416 37 A350G/E367D/T369D 298 339K206A/F217R/N247D/L316D/M322I/A337P/ 420 37 A350G/K362Q/E367N/R373K 299340 K206A/F217R/N247D/Q249H/Q302K/M322I/ 434 40K343G/A350G/E367T/R373K/L397F 234 275 K206A/F217R/N247D/Q302K/A350G/ 41837 E367D/T369D 235 276 K206A/F217R/N247D/Q302K/L316D/ 410 37A337P/A350G/E367D/T369D 236 277 K206A/F217R/Q302K/E367D/T369D 419 37 237278 K206A/F217R/Q302K/L316D/A337P/ 411 37 A350G/E367D/T369D 210 252K206A/F365L/E367N 439 41 211 253 K206A/F365L/E367N/I376V 435 40 212 254K206A/F365L/E367N/R373K/I376V 435 40 238 279K206A/I208V/M322V/K343G/F365L/ 415 37 R373K/I376V 239 280K206A/I208V/R221K/N247D/M322I/ 425 37 K343D/F365L/R373K/I376V 300 341K206A/I208V/R221T/N247D/ 424 38 M322V/K343G/E367N/R373K 214 256K206A/K343D/F365L/E367N 433 41 215 257 K206A/K343G 424 38 216 258K206A/K343G/F365L/E367N/R373K 431 40 240 281 K206A/L269I/P349L/R373K 42842 217 289 K206A/L316D 427 38 218 13 K206A/M322I/E367N/R373K 442 38 301342 K206A/M322I/E367N/R373K 442 38 219 260 K206A/M322I/R373K 435 37 302343 K206A/M322V/K343G/E367N/R373K 425 38 220 261 K206A/M322V/R373K/I376V422 37 221 262 K206A/M390I 414 33 303 344 K206A/N247D/M322I/A337E/K343D/440 40 F365L/E367N/R373K/I376V 241 282K206A/N247D/M322V/K343D/R373K/I376V 415 37 242 283K206A/N247D/M322V/K343G/F365L/R373K 415 37 243 284K206A/N247D/Q302K/A337P/K343G/A350G 417 38 244 285K206A/N247D/Q302K/L316D/A350G 426 38 245 286 K206A/N247D/Q302K/M322V/419 37 F365L/R373K/I376V 222 263 K206A/P228Q/T369D 426 38 223 264K206A/Q302K/A337P/A350G/K362Q 408 38 246 287 K206A/Q302K/L316D/A337P 42138 304 345 K206A/Q302K/L316D/M322I/A337P/ 431 42A350G/K362Q/E367N/T369S/R373K 305 346 K206A/Q302K/L316D/M322I/A337P/ 43842 K343D/E367N/T369S/R373K 224 265 K206A/Q302K/M322V/E367N 441 43 247288 K206A/R221K/N247D/M322V/ 416 37 K343D/R373K 306 347K206A/R221K/N247D/Q302K/ 441 38 M322I/E367N/R373K 307 348K206A/R221K/Q302K/M322I/ 436 38 K343G/E367N/R373K/I376V 226 267K206A/R221T/F365L 427 38 248 289 K206A/R221T/M322V/K343G/R373K 418 37249 290 K206A/R221T/M322V/R373K 423 37 227 268 K206A/R325H 429 38 228269 K206A/R373K 423 37 229 270 K206A/R373K/I376V 422 37 230 271K206A/S374R 433 40 231 272 K206A/T369D 426 38 232 273 K206A/T369S 429 38192 236 K206E 429 38 193 237 K206G 429 38 74 119 K206M 458 44 75 120K206Q 450 38 76 24 K206R 450 38 194 238 K206R 450 38 195 239 K206 S 42938 77 121 K206T/V359S 437 44 78 122 K343D 444 38 79 123 K343G 445 38 80124 K362Q 435 38 81 125 K362R 449 38 82 126 K36D 452 38 83 127 K36E 45038 25 71 K36Q 450 38 84 128 K395* 432 34 85 129 K395G 448 37 86 130K395P 448 37 87 131 K395R 451 38 88 132 K395 S 450 38 89 133 K395T 44837 90 134 K96I 433 36 250 291 K96I/K206A/F217R 412 36 308 349K96I/K206A/F217R/M322I/ 434 40 E367N/T369S/R373K 251 292K96I/K206A/F217R/N247D 411 36 252 293 K96I/K206A/F217R/N247D/ 401 35A350G/E367D/T369D 253 294 K96I/K206A/F217R/N247D/Q302K/L316D/ 393 35A337P/E367D/T369D 309 350 K96I/K206A/F217R/N247D/Q302K/M322I/ 413 36A337P/K343G/A350G/E367N/R373K 310 351 K96I/K206A/N247D/M322I/ 433 40A350G/E367N/T369S/R373K 311 352 K96I/K206A/N247D/Q302K/L316D/M322I/ 42540 A337P/A350G/E367N/T369S/R373K 312 353K96I/K206A/N247D/Q302K/L316D/M322I/ 413 40A337P/A350G/K362Q/E367N/T369S/R373K 91 135 K96L 434 36 92 136 K96R 44337 93 137 K96R/L397V 442 36 94 138 L100F 442 38 313 354L100F/A125S/K206A/I208V/R221K/ 429 38 Q302K/M322I/K343G/E367N/R373K 254295 L100F/K206A 421 38 314 355 L100F/K206A/I208V/N247D/Q302K/ 414 38M322V/K343D/E367N/R373K/I376V 315 356 L100F/K206A/I208V/Q302K/M322V/ 42740 F365L/E367N/R373K/I376V 316 357 L100F/K206A/I208V/R221K/M322V/ 416 38K343D/E367N/R373K 317 358 L100F/K206A/I208V/R221K/M322V/ 422 40K343D/F365L/E367N/R373K 255 296 L100F/K206A/I208V/R221K/N247D/ 417 37Q302K/M322I/K343D/F365L/I376V 256 297 L100F/K206A/I208V/R221K/ 405 37N247D/Q302K/M322V/ K343D/F365L/I376V 318 359L100F/K206A/I208V/R221T/M322V/ 421 38 E367N/R373K/I376V 257 298L100F/K206A/I208V/R221T/N247D/ 405 37 K343D/F365L/I376V 258 299L100F/K206A/I208V/R221T/Q302K/ 420 37 M322I/K343D/I376V 319 360L100F/K206A/M322I/E367N/R373K/I376V 433 38 259 300L100F/K206A/M322V/F365L/R373K/I376V 412 37 260 301L100F/K206A/N247D/F365L/R373K/I376V 411 37 261 302L100F/K206A/N247D/M322V/K343D/I376V 407 37 320 361L100F/K206A/N247D/Q302K/M322I/ 433 38 E367N/R373K 321 362L100F/K206A/R221K/N247D/M322I/ 428 38 K343G/E367N/R373K 262 303L100F/K206A/R221K/N247D/Q302K/ 411 37 M322V/F365L/R373K/I376V 263 304L100F/K206A/R221K/N247D/ 413 37 Q302K/M322V/I376V 264 305L100F/K206A/R221K/N247D/ 407 37 Q302K/M322V/K343D/ R373K/I376V 265 306L100F/K206A/R221K/R373K/I376V 414 37 266 307L100F/K206A/R221T/M322I/K343E/ 419 37 F365L/R373K 267 308L100F/K206A/R221T/N247D/Q302K/ 406 37 K343D/F365L/R373K 322 363L100F/K206A/R221T/Q302K/M322I/ 428 38 K343D/E367N/R373K 268 309L100F/K206A/R373K/I376V 414 37 323 364 L100F/L160I/K206A/R221K/M322V/424 42 E367N/R373K 647 387 L14F/L44R/S47T/Y92H/S166P/ 345 8K206A/F217R/N247D/H271A/ Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392T 95 139 L158A 437 35 96 140 L158I 458 42 97 141 L158M450 40 98 142 L158R 431 35 99 143 L23M 450 38 324 365L23S/K206A/M322I/E367N/R373K 442 38 100 144 L23T 450 38 101 145 L316D448 38 102 146 L316E 448 38 269 310 L37I/K206A/R221K/N247D/M322I/R373K434 37 103 147 L384F 448 35 104 148 L386V 436 31 105 149 L394A 449 37106 150 L394R 450 38 107 151 L394S 450 38 108 152 L394T 449 37 109 153L397* 442 36 110 154 L397D 449 37 111 155 L397H 450 38 112 156 L397I 44937 113 157 L397Q 449 37 114 158 L397R 449 37 115 159 L397T 449 37 116160 L398E 449 37 117 161 L398G 450 38 118 162 L398N 449 37 119 163 L398Q450 38 120 164 L398R 449 37 368 409 L44A/K206A/F217R/N247D/Q302K/L316D/N. D. 36 M322I/A337P/K362Q/E367N/R373K 362 403L44C/K206A/F217R/N247D/Q302K/L316D/ N. D. 32M322I/A337P/K362Q/E367N/R373K 360 401L44E/K206A/F217R/N247D/Q302K/L316D/ N. D. 32M322I/A337P/K362Q/E367N/R373K 374 415L44M/K206A/F217R/N247D/Q302K/L316D/ N. D. 33M322I/A337P/K362Q/E367N/R373K 370 411L44Q/K206A/F217R/N247D/Q302K/L316D/ N. D. 36M322I/A337P/K362Q/E367N/R373K 398 439L44R/A159S/K206A/F217R/N247D/Q302K/ N. D. 34L316D/M322I/A337P/K362Q/E367N/R373K 520 561L44R/A77S/Y92H/K206A/F217R/N247D/ 393 24 Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 270 311 L44R/C143Y/K206A/A337P/A350G 430 38 521 562L44R/D52N/Y92H/K206A/F217R/ 393 24 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 271 312 L44R/E187G/K206A/A337P/A350G 430 38 522563 L44R/E56K/Y92H/K206A/ 393 24 F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 382 423 L44R/H94N/K206A/F217R/N247D/Q302K/N. D. 30 L316D/M322I/A337P/K362Q/E367N/R373K 386 427L44R/H94R/K206A/F217R/N247D/Q302K/ N. D. 34L316D/M322I/A337P/K362Q/E367N/R373K 272 313 L44R/K206A 436 38 273 314L44R/K206A/E367D/T369D 426 37 274 315 L44R/K206A/F217R/A350G 436 38 275316 L44R/K206A/F217R/N247D/A337P 429 38 439 480L44R/K206A/F217R/N247D/H271A/Q302K/ N. D. 36L316D/M322I/A337P/K362Q/E367N/R373K 425 466L44R/K206A/F217R/N247D/H271E/Q302K/ N. D. 36L316D/M322I/A337P/K362Q/E367N/R373K 436 477L44R/K206A/F217R/N247D/H271G/Q302K/ N. D. 36L316D/M322I/A337P/K362Q/E367N/R373K 433 474L44R/K206A/F217R/N247D/H271Q/Q302K/ N. D. 36L316D/M322I/A337P/K362Q/E367N/R373K 452 493L44R/K206A/F217R/N247D/H271R/Q302K/ N. D. 36L316D/M322I/A337P/K362Q/E367N/R373K 430 471L44R/K206A/F217R/N247D/H271T/Q302K/ N. D. 36L316D/M322I/A337P/K362Q/E367N/R373K 441 482L44R/K206A/F217R/N247D/H271V/Q302K/ N. D. 36L316D/M322I/A337P/K362Q/E367N/R373K 435 476L44R/K206A/F217R/N247D/I258L/Q302K/ N. D. 34L316D/M322I/A337P/K362Q/E367N/R373K 450 491L44R/K206A/F217R/N247D/I258M/Q302K/ N. D. 36L316D/M322I/A337P/K362Q/E367N/R373K 442 483L44R/K206A/F217R/N247D/L255A/Q302K/ N. D. 34L316D/M322I/A337P/K362Q/E367N/R373K 429 470L44R/K206A/F217R/N247D/L255C/Q302K/ N. D. 34L316D/M322I/A337P/K362Q/E367N/R373K 424 465L44R/K206A/F217R/N247D/L255E/Q302K/ N. D. 34L316D/M322I/A337P/K362Q/E367N/R373K 443 484L44R/K206A/F217R/N247D/L255S/Q302K/ N. D. 35L316D/M322I/A337P/K362Q/E367N/R373K 449 490L44R/K206A/F217R/N247D/L255T/Q302K/ N. D. 35L316D/M322I/A337P/K362Q/E367N/R373K 432 473L44R/K206A/F217R/N247D/L255V/Q302K/ N. D. 36L316D/M322I/A337P/K362Q/E367N/R373K 440 481L44R/K206A/F217R/N247D/L263C/Q302K/ N. D. 33L316D/M322I/A337P/K362Q/E367N/R373K 437 478L44R/K206A/F217R/N247D/L263E/Q302K/ N. D. 29L316D/M322I/A337P/K362Q/E367N/R373K 445 486L44R/K206A/F217R/N247D/L263F/Q302K/ N. D. 34L316D/M322I/A337P/K362Q/E367N/R373K 427 468L44R/K206A/F217R/N247D/L263G/Q302K/ N. D. 34L316D/M322I/A337P/K362Q/E367N/R373K 447 488L44R/K206A/F217R/N247D/L263W/Q302K/ N. D. 33L316D/M322I/A337P/K362Q/E367N/R373K 276 317L44R/K206A/F217R/N247D/L316D/A337P/ 417 37 A350G/E367D/T369D 277 318L44R/K206A/F217R/N247D/L316D/ 417 37 A337P/E367D/T369D 278 319L44R/K206A/F217R/N247D/L316D/A350G/ 423 37 E367D/T369D 325 366L44R/K206A/F217R/N247D/L316D/M322I/ 422 37 A337P/K343G/K362Q/E367N/R373K446 487 L44R/K206A/F217R/N247D/M259A/Q302K/ N. D. 31L316D/M322I/A337P/K362Q/E367N/R373K 426 467L44R/K206A/F217R/N247D/M259E/Q302K/ N. D. 30L316D/M322I/A337P/K362Q/E367N/R373K 428 469L44R/K206A/F217R/N247D/M259S/Q302K/ N. D. 34L316D/M322I/A337P/K362Q/E367N/R373K 451 492L44R/K206A/F217R/N247D/M259V/Q302K/ N. D. 35L316D/M322I/A337P/K362Q/E367N/R373K 444 485L44R/K206A/F217R/N247D/M259W/Q302K/ N. D. 30L316D/M322I/A337P/K362Q/E367N/R373K 279 320L44R/K206A/F217R/N247D/Q302K/A350G 435 38 326 367L44R/K206A/F217R/N247D/Q302K/L316D/ 427 37 M322I/A337P/K362Q/E367N/R373K513 554 L44R/K206A/F217R/N247D/Q302K/L316D/ N. D. 32M322I/A337P/K362Q/E367N/R373K/D396* 512 553L44R/K206A/F217R/N247D/Q302K/L316D/ N. D. 32M322I/A337P/K362Q/E367N/R373K/K395* 508 549L44R/K206A/F217R/N247D/Q302K/L316D/ N. D. 30M322I/A337P/K362Q/E367N/R373K/L384A 477 518L44R/K206A/F217R/N247D/Q302K/L316D/ N. D. 34M322I/A337P/K362Q/E367N/R373K/L384W 474 515L44R/K206A/F217R/N247D/Q302K/L316D/ N. D. 35M322I/A337P/K362Q/E367N/R373K/L386F 502 543L44R/K206A/F217R/N247D/Q302K/L316D/ N. D. 30M322I/A337P/K362Q/E367N/R373K/L386S 460 501L44R/K206A/F217R/N247D/Q302K/L316D/ N. D. 30M322I/A337P/K362Q/E367N/R373K/L386T 515 556L44R/K206A/F217R/N247D/Q302K/L316D/ N. D. 32M322I/A337P/K362Q/E367N/R373K/L394* 511 552L44R/K206A/F217R/N247D/Q302K/L316D/ N. D. 32M322I/A337P/K362Q/E367N/R373K/L397* 500 541L44R/K206A/F217R/N247D/Q302K/L316D/ N. D. 36M322I/A337P/K362Q/E367N/R373K/M390A 486 527L44R/K206A/F217R/N247D/Q302K/L316D/ N. D. 35M322I/A337P/K362Q/E367N/R373K/M390C 490 531L44R/K206A/F217R/N247D/Q302K/L316D/ N. D. 30M322I/A337P/K362Q/E367N/R373K/M390D 476 517L44R/K206A/F217R/N247D/Q302K/L316D/ N. D. 30M322I/A337P/K362Q/E367N/R373K/M390E 494 535L44R/K206A/F217R/N247D/Q302K/L316D/ N. D. 30M322I/A337P/K362Q/E367N/R373K/M390F 481 522L44R/K206A/F217R/N247D/Q302K/L316D/ N. D. 30M322I/A337P/K362Q/E367N/R373K/M390G 459 500L44R/K206A/F217R/N247D/Q302K/L316D/ N. D. 30M322I/A337P/K362Q/E367N/R373K/M390H 497 538L44R/K206A/F217R/N247D/Q302K/L316D/ N. D. 30M322I/A337P/K362Q/E367N/R373K/M390K 454 495L44R/K206A/F217R/N247D/Q302K/L316D/ N. D. 30M322I/A337P/K362Q/E367N/R373K/M390P 465 506L44R/K206A/F217R/N247D/Q302K/L316D/ N. D. 30M322I/A337P/K362Q/E367N/R373K/M390Q 480 521L44R/K206A/F217R/N247D/Q302K/L316D/ N. D. 30M322I/A337P/K362Q/E367N/R373K/M390R 504 545L44R/K206A/F217R/N247D/Q302K/L316D/ N. D. 32M322I/A337P/K362Q/E367N/R373K/M390S 463 504L44R/K206A/F217R/N247D/Q302K/L316D/ N. D. 34M322I/A337P/K362Q/E367N/R373K/M390T 496 537L44R/K206A/F217R/N247D/Q302K/L316D/ N. D. 32M322I/A337P/K362Q/E367N/R373K/M390V 491 532L44R/K206A/F217R/N247D/Q302K/L316D/ N. D. 30M322I/A337P/K362Q/E367N/R373K/M390W 457 498L44R/K206A/F217R/N247D/Q302K/L316D/ N. D. 34M322I/A337P/K362Q/E367N/R373K/M392A 483 524L44R/K206A/F217R/N247D/Q302K/L316D/ N. D. 34M322I/A337P/K362Q/E367N/R373K/M392C 455 496L44R/K206A/F217R/N247D/Q302K/L316D/ N. D. 35M322I/A337P/K362Q/E367N/R373K/M392D 466 507L44R/K206A/F217R/N247D/Q302K/L316D/ N. D. 35M322I/A337P/K362Q/E367N/R373K/M392E 479 520L44R/K206A/F217R/N247D/Q302K/L316D/ N. D. 30M322I/A337P/K362Q/E367N/R373K/M392F 501 542L44R/K206A/F217R/N247D/Q302K/L316D/ N. D. 30M322I/A337P/K362Q/E367N/R373K/M392G 498 539L44R/K206A/F217R/N247D/Q302K/L316D/ N. D. 34M322I/A337P/K362Q/E367N/R373K/M392I 472 513L44R/K206A/F217R/N247D/Q302K/L316D/ N. D. 35M322I/A337P/K362Q/E367N/R373K/M392K 473 514L44R/K206A/F217R/N247D/Q302K/L316D/ N. D. 33M322I/A337P/K362Q/E367N/R373K/M392L 505 546L44R/K206A/F217R/N247D/Q302K/L316D/ N. D. 30M322I/A337P/K362Q/E367N/R373K/M392N 493 534L44R/K206A/F217R/N247D/Q302K/L316D/ N. D. 30M322I/A337P/K362Q/E367N/R373K/M392P 461 502L44R/K206A/F217R/N247D/Q302K/L316D/ N. D. 35M322I/A337P/K362Q/E367N/R373K/M392Q 478 519L44R/K206A/F217R/N247D/Q302K/L316D/ N. D. 36M322I/A337P/K362Q/E367N/R373K/M392S 507 548L44R/K206A/F217R/N247D/Q302K/L316D/ N. D. 30M322I/A337P/K362Q/E367N/R373K/M392T 484 525L44R/K206A/F217R/N247D/Q302K/L316D/ N. D. 34M322I/A337P/K362Q/E367N/R373K/M392V 485 526L44R/K206A/F217R/N247D/Q302K/L316D/ N. D. 30M322I/A337P/K362Q/E367N/R373K/M392W 503 544L44R/K206A/F217R/N247D/Q302K/L316D/ N. D. 30M322I/A337P/K362Q/E367N/R373K/Q385C 482 523L44R/K206A/F217R/N247D/Q302K/L316D/ N. D. 30M322I/A337P/K362Q/E367N/R373K/Q385G 469 510L44R/K206A/F217R/N247D/Q302K/L316D/ N. D. 36M322I/A337P/K362Q/E367N/R373K/Q385I 462 503L44R/K206A/F217R/N247D/Q302K/L316D/ N. D. 36M322I/A337P/K362Q/E367N/R373K/Q385L 509 550L44R/K206A/F217R/N247D/Q302K/L316D/ N. D. 30M322I/A337P/K362Q/E367N/R373K/Q385T 506 547L44R/K206A/F217R/N247D/Q302K/L316D/ N. D. 30M322I/A337P/K362Q/E367N/R373K/Q385W 514 555L44R/K206A/F217R/N247D/Q302K/L316D/ N. D. 31M322I/A337P/K362Q/E367N/R373K/S393* 492 533L44R/K206A/F217R/N247D/Q302K/L316D/ N. D. 32M322I/A337P/K362Q/E367N/R373K/T389C 475 516L44R/K206A/F217R/N247D/Q302K/L316D/ N. D. 30M322I/A337P/K362Q/E367N/R373K/T389D 487 528L44R/K206A/F217R/N247D/Q302K/L316D/ N. D. 30M322I/A337P/K362Q/E367N/R373K/T389G 489 530L44R/K206A/F217R/N247D/Q302K/L316D/ N. D. 36M322I/A337P/K362Q/E367N/R373K/T389I 499 540L44R/K206A/F217R/N247D/Q302K/L316D/ N. D. 35M322I/A337P/K362Q/E367N/R373K/T389L 456 497L44R/K206A/F217R/N247D/Q302K/L316D/ N. D. 35M322I/A337P/K362Q/E367N/R373K/T389M 488 529L44R/K206A/F217R/N247D/Q302K/L316D/ N. D. 30M322I/A337P/K362Q/E367N/R373K/T389N 495 536L44R/K206A/F217R/N247D/Q302K/L316D/ N. D. 34M322I/A337P/K362Q/E367N/R373K/T389P 468 509L44R/K206A/F217R/N247D/Q302K/L316D/ N. D. 30M322I/A337P/K362Q/E367N/R373K/T389Q 467 508L44R/K206A/F217R/N247D/Q302K/L316D/ N. D. 30M322I/A337P/K362Q/E367N/R373K/T389S 471 512L44R/K206A/F217R/N247D/Q302K/L316D/ N. D. 30M322I/A337P/K362Q/E367N/R373K/T389W 327 368L44R/K206A/F217R/N247D/Q302K/L316D/ 427 37M322I/K343D/A350G/K362Q/E367N/R373K 434 475L44R/K206A/F217R/N247D/R270D/Q302K/ N. D. 36L316D/M322I/A337P/K362Q/E367N/R373K 431 472L44R/K206A/F217R/N247D/R270G/Q302K/ N. D. 36L316D/M322I/A337P/K362Q/E367N/R373K 453 494L44R/K206A/F217R/N247D/R270L/Q302K/ N. D. 36L316D/M322I/A337P/K362Q/E367N/R373K 448 489L44R/K206A/F217R/N247D/R270Q/Q302K/ N. D. 36L316D/M322I/A337P/K362Q/E367N/R373K 280 321L44R/K206A/F217R/Q302K/E367D/T369D 426 37 328 369L44R/K206A/F217R/Q302K/M322I/A337P/ 452 42 A350G/E367N/T369S/R373K 329370 L44R/K206A/I208V/N247D/Q302K/M322I/ 442 38 K343D/E367N/R373K 330 371L44R/K206A/I208V/R221K/M322I/ 443 38 K343D/E367N/R373K 281 322L44R/K206A/I208V/R221K/M322V/ 422 37 K343D/F365L/R373K 331 372L44R/K206A/I208V/R221K/N247D/Q302K/ 441 38 M322I/K343D/E367N/R373K/I376V332 373 L44R/K206A/I208V/R221T/Q302K/M322I/ 449 40K343G/F365L/E367N/R373K/I376V 333 374L44R/K206A/L316D/M322I/A337P/A350G/ 450 42 E367N/T369S/R373K 282 323L44R/K206A/N247D/A337P 429 38 334 375L44R/K206A/N247D/L316D/M322I/A350G/ 443 42 K362Q/E367N/T369S/R373K 283324 L44R/K206A/N247D/Q302K/A337P/ 419 37 A350G/E367D/T369D 335 376L44R/K206A/N247D/Q302K/L316D/M322I/ 432 42 A337P/K343G/A350G/K362Q/E367N/T369S/R373K 336 377 L44R/K206A/N247D/Q302K/M322I/A350G/ 457 42E367N/T369S/R373K 337 378 L44R/K206A/N247D/Q302K/M322I/ 442 38K343D/E367N/R373K 284 325 L44R/K206A/R221T/N247D/M322I/ 432 37K343D/F365L/I376V 285 326 L44R/K96I/K206A 419 36 286 327L44R/K96I/K206A/F217R/N247D 418 36 338 379L44R/K96I/K206A/F217R/N247D/L316D/ 410 35M322I/A337P/A350G/K362Q/E367N/R373K 339 380L44R/K96I/K206A/F217R/N247D/M322I/ 418 35 A350G/K362Q/E367N/R373K 340381 L44R/K96I/K206A/F217R/N247D/M322I/ 428 40A350G/K362Q/E367N/T369S/R373K 341 382 L44R/K96I/K206A/F217R/N247D/M322I/440 40 E367N/T369S/R373K 287 328 L44R/K96I/K206A/F217R/N247D/ 412 36Q302K/A337P/A350G 288 329 L44R/K96I/K206A/F217R/N247D/Q302K/ 397 35A337P/K343D/A350G/E367D/T369D 342 383 L44R/K96I/K206A/F217R/N247D/Q302K/423 36 L316D/M322I/A337P/E367N/R373K 343 384L44R/K96I/K206A/F217R/N247D/Q302K/ 440 40 M322I/E367N/T369S/R373K 344385 L44R/K96I/K206A/F217R/N247D/Q302K/ 418 35 M322I/K362Q/E367N/R373K345 386 L44R/K96I/K206A/F217R/Q219P/ 429 41 N247D/M253K/S266F/D284E/Q290P/L293F/Q302K/V308G/S314F/M322I/ A337P/K343E/E367N/R373K 289 330L44R/K96I/K206A/F217R/Q302K/A350G 419 36 346 387L44R/K96I/K206A/F217R/Q302K/M322I/ 429 40 A350G/K362Q/E367N/T369S/R373K347 388 L44R/K96I/K206A/M322I/A337P/ 435 40 E367N/T369S/R373K 290 331L44R/K96I/K206A/N247D/L316D/A337P/ 400 35 A350G/E367D/T369D 291 332L44R/L100F/K206A/F365L 426 38 292 333L44R/L100F/K206A/I208V/Q219H/N247D/ 416 37 Q302K/M322V/K343D/R373K/I376V348 389 L44R/L100F/K206A/I208V/R221K/M322I/ 442 40K343G/F365L/E367N/R373K 293 334 L44R/L100F/K206A/I208V/R221K/N247D/ 41837 Q302K/M322V/F365L/I376V 349 390 L44R/L100F/K206A/I208V/R221T/N247D/446 40 M322I/F365L/E367N/R373K 350 391L44R/L100F/K206A/I208V/R221T/N247D/ 427 38 M322V/E367N/R373K/I376V 294335 L44R/L100F/K206A/I208V/R221T/ 420 37 N247D/M322V/I376V 295 336L44R/L100F/K206A/I208V/R221T/N247D/ 424 37Q302K/M322I/K343D/F365L/R373K/I376V 351 392L44R/L100F/K206A/I208V/R221T/Q302K/ 440 38 M322I/E367N/R373K/I376V 352393 L44R/L100F/K206A/Q302K/M322I/ 440 38 E367N/R373K/I376V 353 394L44R/L100F/K206A/R221K/M322I/F365L/ 446 40 E367N/R373K/I376V 354 395L44R/L100F/K206A/R221T/M322I/ 447 40 F365L/E367N/R373K 355 396L44R/L100F/K206A/R221T/N247D/M322I/ 433 38 K343D/E367N/R373K/I376V 356397 L44R/L100F/K206A/R221T/N247D/Q302K/ 440 38 M322I/E367N/R373K 357 398L44R/L100F/K206A/R221T/N247D/Q302K/ 427 38 M322V/E367N/R373K/I376V 358399 L44R/L100F/K206A/R221T/Q302K/ 441 38 M322I/E367N/R373K 359 400L44R/L100F/Q181L/K206A/R221T/N247D/ 429 38 Q302K/M322V/E367N/R373K/I376V400 441 L44R/L158C/K206A/F217R/N247D/Q302K/ N. D. 34L316D/M322I/A337P/K362Q/E367N/R373K 405 446L44R/L158E/K206A/F217R/N247D/Q302K/ N. D. 34L316D/M322I/A337P/K362Q/E367N/R373K 422 463L44R/L158G/K206A/F217R/N247D/Q302K/ N. D. 34L316D/M322I/A337P/K362Q/E367N/R373K 407 448L44R/L158H/K206A/F217R/N247D/Q302K/ N. D. 34L316D/M322I/A337P/K362Q/E367N/R373K 396 437L44R/L158M/K206A/F217R/N247D/Q302K/ N. D. 39L316D/M322I/A337P/K362Q/E367N/R373K 414 455L44R/L158Q/K206A/F217R/N247D/Q302K/ N. D. 34L316D/M322I/A337P/K362Q/E367N/R373K 397 438L44R/L158R/K206A/F217R/N247D/Q302K/ N. D. 34L316D/M322I/A337P/K362Q/E367N/R373K 121 165 L44R/L384F 455 35 418 459L44R/N161E/K206A/F217R/N247D/Q302K/ N. D. 34L316D/M322I/A337P/K362Q/E367N/R373K 523 564L44R/N91M/Y92H/K206A/F217R/N247D/ 396 28 Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 524 565 L44R/N91V/Y92H/K206A/F217R/N247D/ 398 27Q302K/L316D/M322I/A337P/K362Q/ E367N/R373K 525 566L44R/Q76H/Y92H/K206A/F217R/ 388 23 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/W368A/R373K 423 464 L44R/R162A/K206A/F217R/N247D/Q302K/ N.D. 35 L316D/M322I/A337P/K362Q/E367N/R373K 416 457L44R/R162G/K206A/F217R/N247D/Q302K/ N. D. 34L316D/M322I/A337P/K362Q/E367N/R373K 410 451L44R/R162H/K206A/F217R/N247D/Q302K/ N. D. 34L316D/M322I/A337P/K362Q/E367N/R373K 406 447L44R/R162K/K206A/F217R/N247D/Q302K/ N. D. 34L316D/M322I/A337P/K362Q/E367N/R373K 421 462L44R/R162Q/K206A/F217R/N247D/Q302K/ N. D. 36L316D/M322I/A337P/K362Q/E367N/R373K 417 458L44R/R162S/K206A/F217R/N247D/Q302K/ N. D. 36L316D/M322I/A337P/K362Q/E367N/R373K 409 450L44R/R165H/K206A/F217R/N247D/Q302K/ N. D. 36L316D/M322I/A337P/K362Q/E367N/R373K 399 440L44R/R165K/K206A/F217R/N247D/Q302K/ N. D. 36L316D/M322I/A337P/K362Q/E367N/R373K 526 567L44R/R74H/Y92H/K206A/F217R/N247D/ 393 24 Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 411 452 L44R/S166A/K206A/F217R/N247D/Q302K/ N. D. 34L316D/M322I/A337P/K362Q/E367N/R373K 415 456L44R/S166D/K206A/F217R/N247D/Q302K/ N. D. 34L316D/M322I/A337P/K362Q/E367N/R373K 419 460L44R/S166E/K206A/F217R/N247D/Q302K/ N. D. 34L316D/M322I/A337P/K362Q/E367N/R373K 404 445L44R/S166F/K206A/F217R/N247D/Q302K/ N. D. 36L316D/M322I/A337P/K362Q/E367N/R373K 403 444L44R/S166G/K206A/F217R/N247D/Q302K/ N. D. 35L316D/M322I/A337P/K362Q/E367N/R373K 412 453L44R/S166H/K206A/F217R/N247D/Q302K/ N. D. 34L316D/M322I/A337P/K362Q/E367N/R373K 402 42L44R/S166P/K206A/F217R/N247D/Q302K/ N. D. 34L316D/M322I/A337P/K362Q/E367N/R373K 408 449L44R/S166R/K206A/F217R/N247D/Q302K/ N. D. 36L316D/M322I/A337P/K362Q/E367N/R373K 420 461L44R/S166T/K206A/F217R/N247D/Q302K/ N. D. 34L316D/M322I/A337P/K362Q/E367N/R373K 363 404L44R/S47D/K206A/F217R/N247D/Q302K/ N. D. 36L316D/M322I/A337P/K362Q/E367N/R373K 375 416L44R/S47I/K206A/F217R/N247D/Q302K/ N. D. 36L316D/M322I/A337P/K362Q/E367N/R373K 365 406L44R/S47N/K206A/F217R/N247D/Q302K/ N. D. 36L316D/M322I/A337P/K362Q/E367N/R373K 623 664L44R/S47N/S166P/K206A/F217R/N247D/ 386 25H271A/A276S/Q302K/L316D/M322I/A337P/ K362Q/E367N/R373K/M392T 624 665L44R/S47N/S166P/K206A/F217R/N247D/ 385 25 H271A/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M390Q 628 668 L44R/S47N/Y92H/S166P/K206A/F217R/ 350 12N247D/H271A/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K/M390H 613 654L44R/S47N/Y92H/S166P/K206A/F217R/ 351 12 N247D/H271A/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M390Q 632 672 L44R/S47N/Y92H/S166P/K206A/F217R/352 12 N247D/H271A/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K/M392T 627667 L44R/S47N/Y92H/S166P/K206A/F217R/ 311 5N247D/M259W/H271A/Q302K/L316D/ M322I/A337P/K362Q/E367N/R373K/M390H/M392T 622 663 L44R/S47N/Y92H/S166P/K206A/ 305 5F217R/N247D/M259W/H271A/ Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M390Q/M392T 615 656 L44R/S47N/Y92H/S166P/K206A/F217R/352 13 N247D/Q302K/L316D/M322I/A337P/K362Q/ E367N/R373K/M390H 361 402L44R/S47R/K206A/F217R/N247D/Q302K/ N. D. 36L316D/M322I/A337P/K362Q/E367N/R373K 631 671L44R/S47T/A53S/Y92H/S166P/K206A/ 344 8F217R/N247D/H271A/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K/M390Q 379420 L44R/S47T/K206A/F217R/N247D/Q302K/ N. D. 32L316D/M322I/A337P/K362Q/E367N/R373K 675 715L44R/S47T/M65V/Y92H/S166P/K206A/ 344 8F217R/N247D/H271A/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K/M392T 657697 L44R/S47T/P67T/Y92H/S166P/K182N/ 338 8K206A/F217R/N247D/H271A/Q302K/L316D/ M322I/A337P/K362Q/E367N/R373K/M392T629 669 L44R/S47T/S166P/K206A/F217R/ 378 21N247D/H271A/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K/M390Q 659 699L44R/S47T/W64L/Y92H/S166P/K206A/ 345 8F217R/N247D/H271A/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K/M392T 672712 L44R/S47T/Y92H/D144Y/S166P/K206A/ 351 8F217R/N247D/H271A/Q302K/L316D/ M322I/A337P/K362Q/E367N/R373K/M392T 646686 L44R/S47T/Y92H/G113C/S166P/K206A/ 345 8F217R/N247D/H271A/Q302K/L316D/ M322I/A337P/K362Q/E367N/R373K/M392T 644684 L44R/S47T/Y92H/S166P/K206A/F217R/ 338 8L237P/N247D/H271A/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K/M392T 687727 L44R/S47T/Y92H/S166P/K206A/ 340 7 F217R/N247D/H271A/Q290R/Q302K/L316D/M322I/A337P/ K362Q/E367N/W368A/R373K/M392T 618 659L44R/S47T/Y92H/S166P/K206A/F217R/ 361 15 N247D/H271A/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 619 660 L44R/S47T/Y92H/S166P/K206A/F217R/ 343 8N247D/H271A/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K/M390H 620 661L44R/S47T/Y92H/S166P/K206A/F217R/ 344 8 N247D/H271A/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M390Q 625 44 L44R/S47T/Y92H/S166P/K206A/F217R/345 8 N247D/H271A/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K/M392T 673713 L44R/S47T/Y92H/S166P/K206A/F217R/ 331 7N247D/H271A/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K/N377Y/M392T 693733 L44R/S47T/Y92H/S166P/K206A/F217R/ 340 7N247D/H271A/Q302K/L316D/M322I/A337P/ K362Q/E367N/W368A/R373K/M392T 682722 L44R/S47T/Y92H/S166P/K206A/F217R/ 348 8N247D/H271A/Q302K/L316Y/M322I/ A337P/K362Q/E367N/R373K/M392T 670 710L44R/S47T/Y92H/S166P/K206A/F217R/ 345 8 N247D/H271A/Q302K/N305L/L316D/M322I/A337P/K362Q/E367N/R373K/M392T 689 729L44R/S47T/Y92H/S166P/K206A/F217R/ 345 8 N247D/H271A/Q302K/N305L/L316D/M322I/A337P/K362Q/E367N/R373K/M392T 684 724 L44R/S47T/Y92H/S166P/K206A/340 7 F217R/N247D/H271A/Q302K/ N305L/L316D/M322I/A337P/K362Q/E367N/W368A/R373K/M392T 678 718 L44R/S47T/Y92H/S166P/K206A/F217R/339 7 N247D/M253W/A257G/H271A/K277R/ Q281L/Q302K/L316D/A319D/M322I/A337P/K362Q/E367N/ R373K/M392T 677 717L44R/S47T/Y92H/S166P/K206A/F217R/ 338 7N247D/M253W/H271A/S273D/P274S/K277R/ Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392T 621 662 L44R/S47T/Y92H/S166P/K206A/F217R/ 307 1N247D/M259E/H271A/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K/M390Q 610651 L44R/S47T/Y92H/S166P/K206A/F217R/ 310 2N247D/M259E/Q302K/L316D/M322I/A337P/ K362Q/E367N/R373K/M390Q 630 670L44R/S47T/Y92H/S166P/K206A/F217R/ 311 1 N247D/M259W/H271A/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M390H 616 657L44R/S47T/Y92H/S166P/K206A/F217R/ 312 1 N247D/M259W/H271A/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M390Q 669 709L44R/S47T/Y92H/S166P/K206A/F217R/ 353 8N247D/P262S/H271A/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K/M392T 614655 L44R/S47T/Y92H/S166P/K206A/F217R/ 363 16 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 692 732 L44R/S47T/Y92H/S166P/K206A/F217R/ 347 8N247D/W256L/H271A/Q302K/L316D/ M322I/A337P/K362Q/E367N/R373K/M392T 666706 L44R/S47T/Y92H/S166P/K206A/F217R/ 345 8P228L/N247D/H271A/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K/M392T 688728 L44R/S47T/Y92H/S166P/K206A/F217R/ 345 8P228Q/N247D/H271A/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K/M392T 674714 L44R/S47T/Y92H/S166P/K206A/F217R/ 345 8P234H/N247D/H271A/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K/M392T 662702 L44R/S47T/Y92H/S166P/K206A/F217R/ 347 8V238I/N247D/H271A/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K/M392T 652692 L44R/S47T/Y92H/S166P/K206A/F217R/ 312 1W246P/N247D/A261G/H271A/Q302K/ N305L/L316D/M322I/A337P/K362Q/E367N/R373K/M392T 655 695 L44R/S47T/Y92H/S166P/K206A/F217R/ 307 0W246P/N247D/A261G/H271A/Q302K/ N305L/L316D/M322I/A337P/K362Q/E367N/W368A/R373K/M392T 645 685 L44R/S47T/Y92H/S166P/P174S/K206A/ 340 8F217R/N247D/H271A/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K/M392T 661701 L44R/S47T/Y92H/S166P/W195C/K206A/ 345 8F217R/N247D/H271A/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K/M392T 366407 L44R/S47V/K206A/F217R/N247D/Q302K/ N. D. 36L316D/M322I/A337P/K362Q/E367N/R373K 401 442L44R/T163S/K206A/F217R/N247D/Q302K/ N. D. 34L316D/M322I/A337P/K362Q/E367N/R373K 388 429L44R/V93L/K206A/F217R/N247D/Q302K/ N. D. 36L316D/M322I/A337P/K362Q/E367N/R373K 389 430L44R/V93S/K206A/F217R/N247D/Q302K/ N. D. 29L316D/M322I/A337P/K362Q/E367N/R373K 387 428L44R/V93T/K206A/F217R/N247D/Q302K/ N. D. 31L316D/M322I/A337P/K362Q/E367N/R373K 385 426L44R/Y92A/K206A/F217R/N247D/Q302K/ N. D. 25L316D/M322I/A337P/K362Q/E367N/R373K 383 424L44R/Y92C/K206A/F217R/N247D/Q302K/ N. D. 24L316D/M322I/A337P/K362Q/E367N/R373K 527 568L44R/Y92E/K206A/F217R/N247D/Q302K/ 377 24L316D/M322I/A337P/K362Q/E367N/R373K 393 434L44R/Y92G/K206A/F217R/N247D/Q302K/ N. D. 24L316D/M322I/A337P/K362Q/E367N/R373K 528 569L44R/Y92H/D130Q/K206A/F217R/N247D/ 393 24 Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 529 570 L44R/Y92H/K182A/K206A/F217R/ 386 24N247D/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K 530 571L44R/Y92H/K182E/K206A/F217R/ 386 24 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 531 572 L44R/Y92H/K82H/K206A/F217R/ 386 24N247D/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K 532 573L44R/Y92H/K182M/K206A/ 386 24 F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 533 574 L44R/Y92H/K182Q/K206A/F217R/ 386 24N247D/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K 534 575L44R/Y92H/K182R/K206A/F217R/ 386 24 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 535 576 L44R/Y92H/K182T/K206A/F217R/ 386 24N247D/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K 536 577L44R/Y92H/K182V/K206A/F217R/ 386 24 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 537 578 L44R/Y92H/K182Y/K206A/F217R/ 386 24N247D/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K 538 579L44R/Y92H/K206A/F217R/N247D/ 392 24 A287C/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 539 580 L44R/Y92H/K206A/F217R/N247D/ 394 24A287H/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K 540 581L44R/Y92H/K206A/F217R/N247D/ 404 24 A287M/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 541 582 L44R/Y92H/K206A/F217R/N247D/ 384 24K283A/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K 542 583L44R/Y92H/K206A/F217R/N247D/ 387 24 K283G/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 543 584 L44R/Y92H/K206A/F217R/N247D/ 385 24K283M/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K 544 585L44R/Y92H/K206A/F217R/N247D/ 385 24 K283V/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 545 586 L44R/Y92H/K206A/F217R/N247D/ 393 24K295A/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K 546 587L44R/Y92H/K206A/F217R/N247D/ 392 24 K295E/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 547 588 L44R/Y92H/K206A/F217R/N247D/ 409 24K295L/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K 548 589L44R/Y92H/K206A/F217R/N247D/ 391 24 K295N/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 549 590 L44R/Y92H/K206A/F217R/N247D/ 393 24K295Q/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K 550 591L44R/Y92H/K206A/F217R/N247D/ 393 24 K295S/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 551 592 L44R/Y92H/K206A/F217R/N247D/ 392 24K295T/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K 552 593L44R/Y92H/K206A/F217R/N247D/ 393 24 Q302K/L316D/A317D/M322I/A337P/K362Q/E367N/R373K 553 594 L44R/Y92H/K206A/F217R/N247D/ 393 24Q302K/L316D/A317Q/M322I/ A337P/K362Q/E367N/R373K 554 595L44R/Y92H/K206A/F217R/N247D/ 388 24 Q302K/L316D/M322I/A337P/A346G/K362Q/E367N/R373K 555 596 L44R/Y92H/K206A/F217R/N247D/ 395 24Q302K/L316D/M322I/A337P/ G344A/K362Q/E367N/R373K 556 597L44R/Y92H/K206A/F217R/N247D/ 388 24 Q302K/L316D/M322I/A337P/G344D/K362Q/E367N/R373K 557 598 L44R/Y92H/K206A/F217R/N247D/ 388 24Q302K/L316D/M322I/A337P/ G344S/K362Q/E367N/R373K 558 599L44R/Y92H/K206A/F217R/N247D/ 391 24 Q302K/L316D/M322I/A337P/I353L/K362Q/E367N/R373K 559 600 L44R/Y92H/K206A/F217R/N247D/ 386 23Q302K/L316D/M322I/A337P/ K362Q/E367N/L372W/R373K 395 40L44R/Y92H/K206A/F217R/N247D/ N. D. 24 Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 560 601 L44R/Y92H/K206A/F217R/N247D/ 388 23Q302K/L316D/M322I/A337P/ K362Q/E367N/W368A/R373K 561 602L44R/Y92H/K206A/F217R/N247D/ 412 31 Q302K/L316D/M322I/A337P/K362Q/E367N/W368L/R373K 562 603 L44R/Y92H/K206A/F217R/N247D/ 393 24Q302K/L316D/M322I/A337P/ K362Q/E367N/W368N/R373K 563 604L44R/Y92H/K206A/F217R/N247D/ 400 28 Q302K/L316D/M322I/A337P/K362Q/E367N/W368R/R373K 564 605 L44R/Y92H/K206A/F217R/N247D/ 407 29Q302K/L316D/M322I/A337P/ K362Q/E367N/W368V/R373K 565 606L44R/Y92H/K206A/F217R/N247D/ 393 24 Q302K/L316D/M322I/A337P/N348E/K362Q/E367N/R373K 566 607 L44R/Y92H/K206A/F217R/N247D/ 393 24Q302K/L316D/M322I/A337P/ N348M/K362Q/E367N/R373K 567 608L44R/Y92H/K206A/F217R/N247D/ 393 24 Q302K/L316D/M322I/A337P/N348Q/K362Q/E367N/R373K 568 609 L44R/Y92H/K206A/F217R/N247D/ 393 24Q302K/L316D/M322I/A337P/ N348R/K362Q/E367N/R373K 569 610L44R/Y92H/K206A/F217R/N247D/ 393 24 Q302K/L316D/M322I/A337P/N348W/K362Q/E367N/R373K 570 611 L44R/Y92H/K206A/F217R/N247D/ 391 24Q302K/L316D/M322I/A337P/ T354S/K362Q/E367N/R373K 571 612L44R/Y92H/K206A/F217R/N247D/ 400 24 Q302K/N305K/L316D/M322I/A337P/K362Q/E367N/R373K 572 613 L44R/Y92H/K206A/F217R/N247D/ 393 24Q302K/N305L/L316D/M322I/ A337P/K362Q/E367N/R373K 573 614L44R/Y92H/K206A/F217R/N247D/ 393 24 Q302K/S314A/L316D/M322I/A337P/K362Q/E367N/R373K 574 615 L44R/Y92H/K206A/F217R/N247D/ 395 24Q302K/S314H/L316D/M322I/ A337P/K362Q/E367N/R373K 575 616L44R/Y92H/K206A/F217R/N247D/ 388 24 Q302K/S314N/L316D/M322I/A337P/K362Q/E367N/R373K 576 617 L44R/Y92H/K206A/F217R/N247D/ 388 24Q302K/S314Y/L316D/M322I/ A337P/K362Q/E367N/R373K 577 618L44R/Y92H/K206A/F217R/W246A/ 395 24 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 578 619 L44R/Y92H/K206A/F217R/W246I/ 399 24N247D/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K 579 620L44R/Y92H/K206A/F217R/W246P/ 387 24 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 580 621 L44R/Y92H/K206A/F217R/W246R/ 396 24N247D/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K 581 622L44R/Y92H/K206A/F217R/W246S/ 402 24 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 582 623 L44R/Y92H/K206A/S210A/F217R/ 393 24N247D/Q302K/L316D/M322I/ A337P/A350T/K362Q/E367N/R373K 583 624L44R/Y92H/K206A/S210A/F217R/ 393 24 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 584 625 L44R/Y92H/K206A/S210E/F217R/ 393 24N247D/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K 585 626L44R/Y92H/K206A/S210K/F217R/ 407 24 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 586 627 L44R/Y92H/K206A/S210N/F217R/ 393 24N247D/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K 587 628L44R/Y92H/K206A/S210R/F217R/ 408 24 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 588 629 L44R/Y92H/K96A/K206A/F217R/ 380 24N247D/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K 589 630L44R/Y92H/K96W/K206A/F217R/ 399 26 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 617 658 L44R/Y92H/L136V/S166P/ 347 8K206A/F217R/N247D/M259A/Q302K/ L316D/M322I/A337P/K362Q/E367N/R373K/M390Q 590 631 L44R/Y92H/P179M/K206A/F217R/ 414 29N247D/Q302K/L316D/ M322I/A337P/K362Q/E367N/R373K 591 632L44R/Y92H/R189K/K206A/F217R/ 390 24 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 592 633 L44R/Y92H/R189V/K206A/F217R/ 398 24N247D/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K 626 666L44R/Y92H/S166P/K206A/F217R/ 358 13 N247D/H271A/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M390Q 611 652 L44R/Y92H/S166P/K206A/F217R/ 36014 N247D/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K/M390Q 612 953L44R/Y92H/S166P/K206A/F217R/ 361 14 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392T 593 634 L44R/Y92H/S95A/K206A/F217R/ 396 27N247D/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K 594 635L44R/Y92H/S95E/K206A/F217R/ 375 24 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 595 636 L44R/Y92H/T186A/K206A/F217R/ 393 24N247D/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K 596 637L44R/Y92H/T186G/K206A/F217R/ 393 24 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 597 638 L44R/Y92H/T186V/K206A/F217R/ 401 24N247D/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K 598 639L44R/Y92H/Y120H/K206A/F217R/ 393 24 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 599 640 L44R/Y92H/Y120S/K206A/F217R/ 393 24N247D/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K 600 641L44R/Y92H/Y120S/K206A/F217R/ 388 24 N247D/Q302K/L316D/M322I/A337P/L341F/K362Q/E367N/R373K 380 421 L44R/Y92K/K206A/F217R/N247D/ N. D.24 Q302K/L316D/M322I/A337P/ K362Q/E367N/R373K 390 431L44R/Y92Q/K206A/F217R/N247D/ N. D. 24 Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 394 435 L44R/Y92R/K206A/F217R/N247D/ N. D. 30Q302K/L316D/M322I/A337P/ K362Q/E367N/R373K 381 422L44R/Y92S/K206A/F217R/N247D/ N. D. 24 Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 392 433 L44R/Y92T/K206A/F217R/N247D/ N. D. 24Q302K/L316D/M322I/A337P/ K362Q/E367N/R373K 384 425L44R/Y92V/K206A/F217R/N247D/ N. D. 35 Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 369 410 L44S/K206A/F217R/N247D/Q302K/ N. D. 36L316D/M322I/A337P/K362Q/E367N/R373K 122 166 L44T 456 37 378 419L44T/K206A/F217R/N247D/Q302K/L316D/ N. D. 36M322I/A337P/K362Q/E367N/R373K 372 413L44V/K206A/F217R/N247D/Q302K/L316D/ N. D. 36M322I/A337P/K362Q/E367N/R373K 371 412 L44W/K206A/F217R/N247D/Q302K/ N.D. 32 L316D/M322I/A337P/K362Q/E367N/R373K 123 167 M20D/Q302K 450 38 124168 M253F 444 38 125 169 M322I 462 38 126 170 M390D 425 31 127 171 M390R430 31 128 172 M390T 438 35 129 173 M392G 435 31 130 174 M392P 433 31131 175 M392S 448 37 601 642 M39C/L44R/Y92H/K206A/F217R/ 367 19N247D/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K 683 723M39E/E43D/L44R/S47T/Y92H/S166P/ 309 6 K206A/F217R/W246P/N247D/M253W/H271A/S273D/Q302K/L316D/M322I/A337P/ K362Q/E367N/W368A/R373K/M392T 660700 M39E/L44R/S47T/Y92H/S166P/K206A/ 302 1F217R/N247D/A261G/H271A/Q302K/ N305L/L316D/M322I/A337P/K362Q/E367N/R373K/M392T 668 708 M39E/L44R/S47T/Y92H/S166P/K206A/ 329 8F217R/N247D/H271A/Q302K/L316D/ M322I/A337P/K362Q/E367N/ R373K/M392T 676716 M39E/L44R/S47T/Y92H/S166P/K206A/ 324 7F217R/N247D/H271A/Q302K/L316D/ M322I/A337P/K362Q/E367N/W368A/R373K/M392T 639 679 M39E/L44R/S47T/Y92H/S166P/K206A/ 343 8F217R/N247Y/H271A/Q302K/L316D/ M322I/A337P/K362Q/E367N/R373K/M392T 658698 M39E/L44R/S47T/Y92H/S166P/K206A/ 323 8F217R/W246P/N247D/H271A/Q302K/ L316D/M322I/A337P/K362Q/E367N/R373K/M392T 602 643 M39E/L44R/Y92H/K206A/F217R/ 363 19N247D/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K 364 405M39H/L44R/K206A/F217R/N247D/ N. D. 32 Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 367 408 M39R/L44R/K206A/F217R/N247D/ N. D. 32Q302K/L316D/M322I/A337P/ K362Q/E367N/R373K 603 644M39R/L44R/Y92H/K206A/F217R/ 368 19 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 377 418 M39T/L44R/K206A/F217R/N247D/ N. D. 32Q302K/L316D/M322I/A337P/ K362Q/E367N/R373K 604 645M39V/L44R/Y92H/K206A/F217R/ 393 24 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 132 176 M39Y 451 37 376 417M41P/L44R/K206A/F217R/N247D/ N. D. 35 Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 373 414 M41R/L44R/K206A/F217R/N247D/ N. D. 36Q302K/L316D/M322I/A337P/ K362Q/E367N/R373K 133 177 N388R 454 38 134 178N91Q 438 32 26 72 P179S/R373K 430 37 135 179 Q190S/T369D 448 38 136 180Q249A 449 38 27 73 Q299R/M322V/R373K 451 37 28 74 Q299R/Q302K/R373K 45137 29 75 Q299R/Q302K/R373K/I376V 450 37 137 181 Q302A 450 38 30 76Q302K/I376V 443 37 138 182 Q385H 435 32 139 183 Q385I 447 38 140 184Q385L 445 38 141 185 Q391G 449 36 142 186 Q80A 450 38 143 187 Q80H 45038 144 188 Q80V 459 38 145 189 Q88A 448 38 146 190 Q88F 456 38 147 191Q88H 448 38 148 192 Q88R 448 38 149 193 Q88S 448 38 150 194 R162H 446 35151 195 R162S 450 37 225 226 R165S/K206A 427 39 152 196 R221K/A350G 45038 153 197 R221T 450 38 154 198 R301I/K362T 449 41 155 199 R301L 450 38156 200 R371S 456 39 157 201 R371V 452 40 31 77 R373K 444 37 32 78R373K/I376V 443 37 665 705 R7C/L44R/S47T/Y92H/S166P/K206A/ 340 7F217R/N247D/H271A/Q302K/L316D/ M322I/A337P/K362Q/E367N/W368A/R373K/M392T 650 690 R7H/T10P/L44R/S47T/Y92H/S166P/ 345 8K206A/F217R/N247D/H271A/Q302K/ L316D/M322I/A337P/K362Q/E367N/R373K/M392T 681 721 R7P/L44R/S47T/Y92H/S166P/K206A/ 345 8F217R/N247D/H271A/Q302K/L316D/ M322I/A337P/K362Q/E367N/R373K/M392T 654694 R7S/L44R/S47T/Y92H/S166P/K206A/ 345 8 F217R/N247D/H271A/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392T 158 202 R87K 435 32 159 203R87P/L398R 423 31 160 204 S166A 440 35 161 205 S166H 447 35 162 206S166K 441 35 163 207 S31D 450 38 164 208 S34D/M392P 439 31 165 209 S34G450 38 166 210 S34H/M390R 430 31 167 211 S34R 450 38 168 212 S374M 45440 169 213 S374T 439 37 170 214 S393E 447 37 171 215 S393G 447 37 172216 S393H 454 38 173 217 S393P 452 37 174 218 S47I 450 38 175 219 S47R459 38 176 220 S47T 433 33 177 221 S95D 422 31 178 222 S95E 414 31 179223 S95Q 446 36 686 728 T10P/E17G/L44R/S47T/Y92H/S166P/ 352 8K206A/F217R/N247D/H271A/Q302K/ L316D/M322I/A337P/K362Q/E367N/R373K/M392T 642 682 T10P/E43D/L44R/S47T/Y92H/S166P/ 315 1K206A/F217R/N247D/A261G/H271A/ Q302K/N305L/L316D/M322I/A337P/K362Q/E367N/W368A/R373K/M392T 690 730 T10P/L44R/S47T/Y92H/M156V/S166P/ 333 8K206A/F217R/N247D/H271A/Q302K/ L316D/M322I/A337P/K362Q/E367N/R373K/M392T 638 678 T10P/L44R/S47T/Y92H/S166P/K206A/ 318 1F217R/N247D/A261G/H271A/Q302K/ L316D/M322I/A337P/K362Q/E367N/R373K/M392T 651 691 T10P/L44R/S47T/Y92H/S166P/K206A/ 345 8F217R/N247D/H271A/Q302K/L316D/ M322I/A337P/K362Q/E367N/R373K/M392T 691731 T10P/L44R/S47T/Y92H/S166P/K206A/ 345 8F217R/N247D/H271A/Q302K/L316D/ M322I/A337P/K362Q/E367N/R373K/M392T 671711 T10P/L44R/S47T/Y92H/S166P/K206A/ 340 7F217R/N247D/H271A/Q302K/L316D/ M322I/A337P/K362Q/E367N/W368A/R373K/M392T 643 683 T10P/L44R/S47T/Y92H/S166P/K206A/ 335 8F217R/N247D/H271A/Q302K/L316D/ M322I/R325S/A337P/K362Q/E367N/R373K/M392T 664 704 T10P/L44R/S47T/Y92H/S166P/K206A/ 312 2F217R/N247D/Q252H/M253R/A254E/ A261G/H271A/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392T 656 696 T10P/L44R/S47T/Y92H/S166P/K206A/312 1 F217R/W246P/N247D/A261G/H271A/ Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392T 653 693 T10P/L44R/S47T/Y92H/S166P/K206A/ 339 8F217R/W246P/N247D/H271A/Q302K/ L316D/M322I/A337P/K362Q/E367N/R373K/M392T 605 646 T10P/L44R/Y92H/K206A/F217R/N247D/ 393 24Q302K/L316D/M322I/A337P/ K362Q/E367N/R373K 606 647T10P/L44R/Y92H/R189L/K206A/F217R/ 395 24 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 640 680 T10P/M39E/E43D/L44R/S47T/Y92H/ 329 8S166P/K206A/F217R/N247D/H271A/ Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392T 648 46 T10P/M39E/L44R/S47T/Y92H/S166P/ 297 0K206A/F217R/N247D/A261G/H271A/ Q302K/L316D/M322I/A337P/K362Q/E367N/W368A/R373K/M392T 680 720 T10P/M39E/L44R/S47T/Y92H/S166P/ 329 8K206A/F217R/N247D/H271A/Q302K/ L316D/M322I/A337P/K362Q/E367N/R373K/M392T 679 719 T10P/M39E/L44R/S47T/Y92H/S166P/ 329 8K206A/F217R/N247D/H271A/Q302K/ N305L/L316D/M322I/A337P/K362Q/E367N/R373K/M392T 641 681 T10P/M39E/L44R/S47T/Y92H/S166P/ 313 8K206A/F217R/N247D/S266P/H271A/ Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392T 649 689 T10P/M39E/L44R/S47T/Y92H/S166P/ 323 8K206A/F217R/W246P/N247D/H271A/ Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392T 180 224 T369D 447 38 181 225 T369S 450 38 182 226T389S 436 31 607 648 T8L/L44R/Y92H/K206A/F217R/ 398 24N247D/Q302K/L316D/M322I/ A337P/K362Q/E367N/R373K 608 649T8Q/L44R/Y92H/K206A/F217R/ 393 24 N247D/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K 183 227 V133I 457 38 184 228 V168A 434 37 185229 V168L 445 38 186 230 V345N 447 38 187 231 V345Y 449 38 188 232 V359E429 38 189 233 V93I 443 37 190 234 W178H 448 38 191 235 W178S 442 38N.D.-Not determined.

While the invention has been described with reference to the specificembodiments, various changes can be made and equivalents can besubstituted to adapt to a particular situation, material, composition ofmatter, process, process step or steps, thereby achieving benefits ofthe invention without departing from the scope of what is claimed.

For all purposes in the United States of America, each and everypublication and patent document cited in this application isincorporated herein by reference as if each such publication or documentwas specifically and individually indicated to be incorporated herein byreference. Citation of publications and patent documents is not intendedas an indication that any such document is pertinent prior art, nor doesit constitute an admission as to its contents or date.

What is claimed is:
 1. A recombinant alpha galactosidase A and/orbiologically active recombinant alpha galactosidase A fragmentcomprising an amino acid sequence comprising at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99% sequence identity to SEQID NO:5.
 2. The recombinant alpha galactosidase A of claim 1, whereinsaid alpha galactosidase A comprises at least one mutation in at leastone position as provided in Tables 2.1, 2.2, 2.4, 2.5, 2.6, 2.7, 2.8,2.9, and/or 7.1, wherein the positions are numbered with reference toSEQ ID NO:5.
 3. The recombinant alpha galactosidase A of claim 2,wherein said alpha galactosidase A comprises at least one mutation in atleast one position as provided in Table 2.3, wherein the positions arenumbered with reference to SEQ ID NO:10.
 4. The recombinant alphagalactosidase A of any of claims 1-3, wherein said recombinant alphagalactosidase A is derived from a human alpha galactosidase A.
 5. Arecombinant alpha galactosidase A comprising the polypeptide sequence ofSEQ ID NO:15, 13, 10, 18, 40, 42, 44, or
 46. 6. The recombinant alphagalactosidase A of any of claims 1-5, wherein said recombinant alphagalactosidase A is more thermostable than the alpha galactosidase A ofSEQ ID NO:5.
 7. The recombinant alpha galactosidase A of any of claims1-6, wherein said recombinant alpha galactosidase A is more stable at pH7.4 than the alpha galactosidase A of SEQ ID NO:5.
 8. The recombinantalpha galactosidase A of claim 7, wherein said recombinant alphagalactosidase A is more stable at pH 4.3 than the alpha galactosidase Aof SEQ ID NO:5.
 9. The recombinant alpha galactosidase A of claim 7,wherein said recombinant alpha galactosidase A is more stable toexposure to serum than the alpha galactosidase A of SEQ ID NO:5.
 10. Therecombinant alpha galactosidase A of any of claims 1-9, wherein saidrecombinant alpha galactosidase A is a deimmunized alpha galactosidaseA.
 11. The recombinant alpha galactosidase A of any of claims 1-10,wherein said recombinant alpha galactosidase A is a deimmunized alphagalactosidase A provided in Table 7.1.
 12. The recombinant alphagalactosidase A of any of claims 1-11, wherein said recombinant alphagalactosidase A is purified.
 13. The recombinant alpha galactosidase Aof any of claims 1-12, wherein said recombinant alpha galactosidase Aexhibits at least one improved property selected from: i) enhancedcatalytic activity; ii) increased tolerance to pH 7.4; iii) increasedtolerance to pH 4.3; iv) increased tolerance to serum; or v) reducedimmunogenicity; or a combination of any of i), ii), iii), iv), or v), ascompared to a reference sequence.
 14. The recombinant alphagalactosidase A of claim 13, wherein said reference sequence is SEQ IDNO:5 or SEQ ID NO:10.
 15. A composition comprising at least onerecombinant alpha galactosidase A of any of claims 1-14.
 16. Arecombinant polynucleotide sequence encoding at least one recombinantalpha galactosidase A set forth in any of claims 1-15.
 17. Therecombinant polynucleotide sequence of claim 16, wherein saidpolynucleotide sequence is codon-optimized.
 18. An expression vectorcomprising the recombinant polynucleotide sequence of claim 16 and/or17.
 19. The expression vector of claim 18, wherein said recombinantpolynucleotide sequence is operably linked to a control sequence. 20.The expression vector of claim 19, wherein said control sequence is apromoter.
 21. The expression vector of claim 20, wherein said promoteris a heterologous promoter.
 22. A host cell comprising the expressionvector of any of claims 18-21.
 23. The host cell of claim 22, whereinsaid host cell is eukaryotic.
 24. A method of producing an alphagalactosidase A variant, comprising culturing said host cell of claim 22or 23, under conditions that said alpha galactosidase A encoded by saidrecombinant polynucleotide is produced.
 25. The method of claim 24,further comprising the step of recovering said alpha galactosidase A.26. The method of claim 25, further comprising the step of purifyingsaid alpha galactosidase A.
 27. A pharmaceutical composition for thetreatment of Fabry disease, comprising the enzyme composition of claim15.
 28. The pharmaceutical composition of claim 27, further comprising apharmaceutically acceptable carrier and/or excipient.
 29. Thepharmaceutical composition of claims 27 and/or 28, wherein saidcomposition is suitable for parenteral injection or infusion to a human.30. A method for treating and/or preventing the symptoms of Fabrydisease in a subject, comprising providing a subject having Fabrydisease, and providing the pharmaceutical composition of any of claims27-39, to said subject.
 31. The method of claim 30, wherein saidsymptoms of Fabry disease are ameliorated.
 32. The method of claim 30and/or 31, wherein said subject is able to eat a diet that is lessrestricted in its fat content than diets required by subjects exhibitingthe symptoms of Fabry disease.
 33. The method of any of claims 30-32,wherein said subject is an infant or child.
 34. The method of any ofclaims 30-32, wherein said subject is an adult or young adult.
 35. Useof the compositions provided in any of claims 15, and 27-29.